Cells In G1 Phase Research Articles

Astrocyte-specific hypoxia-inducible factor 1 (HIF-1) does not disrupt the endothelial barrier during hypoxia in vitro

BackgroundAstrocytes (AC) are essential for brain homeostasis. Much data suggests that AC support and protect the vascular endothelium, but increasing evidence indicates that during injury conditions they may lose their supportive role resulting in endothelial cell activation and BBB disturbance. Understanding the triggers that flip this switch would provide invaluable information for designing new targets to modulate the brain vascular compartment. Hypoxia-inducible factor-1 (HIF-1) has long been assumed to be a culprit for barrier dysfunction as a number of its target genes are potent angiogenic factors. Indeed AC themselves, reservoirs of an array of different growth factors and molecules, are frequently assumed to be the source of such molecules although direct supporting evidence is yet to be published. Being well known reservoirs of HIF-1 dependent angiogenic molecules, we asked if AC HIF-1 dependent paracrine signaling drives brain EC disturbance during hypoxia.MethodsFirst we collected conditioned media from control and siRNA-mediated HIF-1 knockdown primary rat AC that had been exposed to normoxic or hypoxic conditions. The conditioned media was then used to culture normoxic and hypoxic (1% O2) rat brain microvascular EC (RBE4) for 6 and 24 h. Various activation parameters including migration, proliferation and cell cycling were assessed and compared to untreated controls. In addition, tight junction localization and barrier stability per se (via permeability assay) was evaluated.ResultsAC conditioned media maintained both normoxic and hypoxic EC in a quiescent state by suppressing EC metabolic activity and proliferation. By FACs we observed reduced cell cycling with an increased number of cells in G0 phase and reduced cell numbers in M phase compared to controls. EC migration was also blocked by AC conditioned media and in correlation hypoxic tight junction organization and barrier functionality was improved. Surprisingly however, AC HIF-1 deletion did not impact EC responses or barrier stability during hypoxia.ConclusionsThis study demonstrates that AC HIF-1 dependent paracrine signaling does not contribute to AC modulation of EC barrier function under normoxic or hypoxic conditions. Thus other cell types likely mediate EC permeability in stress scenarios. Our data does however highlight the continuous protective effect of AC on the barrier endothelium. Exploring these protective mechanisms in more detail will provide essential insight into ways to prevent barrier disturbance during injury and disease.

Who gets a license: DNA synthesis in quiescent cells re-entering the cell cycle.

The precise regulation of the entry into S phase is critical for preventing genome instability. The first step in the initiation of eukaryotic DNA synthesis occurs in G1 phase cells and involves the loading of the conserved MCM helicase onto multiple origins of replication in a process known as origin licensing. In proliferating metazoan cells, an origin-licensing checkpoint delays initiation until high levels of MCM loading occur, with excess origins being licensed. One function of this checkpoint is to ensure that S phase can be completed in the face of replication stress by activation of dormant MCM bound origins. However, when both metazoan and yeast cells enter S phase from quiescence or G0 phase, a non-growing but reversible cell cycle state, origins are significantly under-licensed. In metazoan cells, under-licensing is the result of a compromised origin-licensing checkpoint. In budding yeast, our study has revealed that under-licensing can be attributed to the chromatin structure at a class of origins that is inhibitory to the binding of MCM. Thus, defects in multiple pathways may contribute to the failure to fully license origins in quiescent cells re-entering the cell cycle, thereby promoting a higher risk of genome instability.

Prolonged cetuximab treatment promotes p27Kip1-mediated G1 arrest and autophagy in head and neck squamous cell carcinoma

Cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is an efficient anti-tumor therapeutic agent that inhibits the activation of EGFR; however, data related to the cellular effects of prolonged cetuximab treatment are limited. In this study, the long-term cellular outcome of prolonged cetuximab treatment and the related molecular mechanism were explored in a head and neck squamous cell carcinoma cell line constitutively expressing a fluorescent ubiquitination-based cell cycle indicator. Fluorescent time-lapse imaging was used to assess clonal growth, cell motility, and cell-cycle progression. Western blot analysis was performed to measure the level of phosphorylation and protein-expression following cetuximab treatment. Over 5 days cetuximab treatment decreased cell motility and enhanced G1 phase cell arrest in the central region of the colonies. Significantly decreased phosphorylation of retinoblastoma, Skp2, and Akt-mTOR proteins, accumulation of p27Kip1, and induction of type II LC3B were observed over 8 days cetuximab treatment. Results of the present study elucidate the cetuximab-dependent inhibition of cell migration, resulting in high cell density-related stress and persistent cell-cycle arrest at G1 phase culminating in autophagy. These findings provide novel molecular insights related to the anti-tumor effects of prolonged cetuximab treatment with the potential to improve future therapeutic strategy.

DNAJC5 promotes hepatocellular carcinoma cells proliferation though regulating SKP2 mediated p27 degradation

DNAJC5 (DnaJ heat shock protein family (Hsp40) member C5), also known as cysteine tandem protein (CSPα), is important for maintaining the normal function of nerve tissues, but its oncogenic function remains unknown. Here, we report a unique mechanism underlying the oncogenic function of DNAJC5. DNAJC5 protein expression is highly detectable in human hepatocellular carcinoma (HCC) tissues and is strongly related to a poor prognosis among HCC patients. DNAJC5 overexpression promotes HCC cell proliferation and reduced the ratio of cells in G1 phase of the cell cycle. Furthermore, DNAJC5 interacts with SKP2 and enhances the degradation of p27 (a cyclin-dependent kinase inhibitor1B) by promoting formation of the SKP2-p27 complex. In contrast, DNAJC5 knockdown rescues the SKP2-mediated decrease in p27 protein levels. These results reveal that the DNAJC5-SKP2-p27 pathway is a novel mechanism for the oncogenic function of DNAJC5 in HCC.

Dioscorea deltoidea Leaf Extract (DDLE) Targets PI3K/AKT/mTOR Pathway and Inhibits Ovarian Cancer Cell Growth.

Ovarian cancer is the malignant tumour of the female reproductive organ with highest mortality rate among all the types of gynaecological tumours. This study investigated the effect of Dioscorea deltoidea leaf extract (DDLE) on OV-90 and CAOV4 ovarian cancer cells. The results demonstrated that DDLE suppresses OV-90 and CAOV3 cell viability significantly in dose dependent manner. The OV-90 and CAOV3 cell viability were reduced to 24 and 27% respectively with 20 mg/mL DDLE treatment. Five mg/mL DDLE treatment of OV-90 and CAOV4 cells raised percentage of cells in G2-phase to 55.9 and 51.2%, respectively. In 5mg/mL DDLE -treated OV-90 and CAOV4 cells a prominent suppression in cyclin-D1 and cyclin B1 proteins was observed in 48 h. The DDLE treatment promoted OV-90 and CAOV3 cell apoptosis to 34.65 and 29.89%, respectively. The Fas, FasL, cleaved caspase-3, and Bax levels were up-regulated markedly in the cells after DDLE treatment. Moreover, DDLE treatment suppressed p-mTOR, p-AKT and p-PI3K expression in OV-90 and CAOV3 cells. Thus, DDLE suppressed ovary cancer cell viability and elevated cell apoptosis. Inhibitory effect of DDLE on ovarian cancer cells is associated with targeting PI3K/AKT/mTOR pathway.

DNA BINDING, IN VITRO CYTOTOXICITY AND ANTICANCER DRUG MECHANISM OF COPPER(II) COMPLEX CONTAINING PYRIDYL-TRIAZINE LIGAND

Mononuclear copper(II) complex [Cu(dppt)2(H2O)2](ClO4)2 (1), where dppt is bidentate NpyNtz donor asymmetric ligand (pyridyl-triazine) has been isolated. The X-ray crystal structure of 1 possesses a CuN4O2 chromophore with elongated octahedral geometry. The electronic and EPR spectral properties demonstrate that the solvent molecules strongly interacted in the axial position thereby weakens the CuN4 coordination plane. Absorption and emission spectral and electrochemical measurements clearly show the partial intercalative binding of 1 to calf thymus (CT) DNA. Remarkably, it exhibits potent cytotoxicity (IC50, 3.73 M) against human cervical carcinoma cells (HeLa), which is 4.5 times better than cisplatin and is non-toxic (IC50, >500 M) to normal mouse embryonic fibroblasts cells (NIH 3T3). It blocks cell cycle progression of HeLa cells in G1 phase. FACSverse analysis of 1 is suggestive of ROS (reactive oxygen species) generation and absolutely induces apoptotic cell death in HeLa cells.

A multivariate comparative clinical pharmacotherapeutic efficacy and chronopharmacovigilance assessment study of ofloxacin, one of the commonplace, TGFβ1 inducing and telomerase impairing fluoroquinolones, in treating acute gastroenteritis, chronic obstructive pulmonary disease, new drug-sensitive tuberculosis, recurrent mixed cutaneous infections, and post-surgical refractory wound infections, among the global patients, with heterogenous pharmacogeographic and pharmacogenomic constitution

Background: Ofloxacin has an inhibitory effect on DNA gyrase, DNA topoisomerase IV and IL-1α, IL-6, IL-8, TNFa; and a superinducing effect on IL-2. Ofloxacin has profound bactericidal, anti-tubercular, anti-leprotic, anti-viral including anti-coronavirus, anti-fungal, anti-protozoal, comedolytic, anti-comedogenic, anti-inflammatory, immunomodulatory, and anti-malignant: pro-apoptotic and anti-proliferative potential, including TGFb1 targeted G2 phase cell cycle arrest and telomerase activity impairment. Objectives of the study were a comparative clinical pharmacotherapeutic efficacy and chronopharmacovigilance assessment study, of ofloxacin, one of the commonplace TGFb1 inducing and telomerase impairing fluoroquinolones, in treating heterogenous global patients, suffering from different diseases.Methods: A prospective, multivariate study of 100 patients, allotted into group A (acute gastroenteritis) =20, group B (chronic obstructive pulmonary disease) =20, group C (new drug-sensitive tuberculosis) =20, group D (recurrent mixed cutaneous infections) =20, and group E (post-surgical refractory wound infections) =20, was prescribed ofloxacin 200-400 mg twice daily, according to required prescribed regimens. A comparative pharmacotherapeutic efficacy assessment was made from the complete recovery time-periods, including the residual recovery time-periods. The chronopharmacovigilance assessment was made by adverse effects occurrence monitoring during treatment period or follow-up, with an Adverse Event Case Report Form. Results: The residual recovery time-periods, in group A=0 days, group B=2 days, group E=3 days, group D=3 days, and group C=7 days. Adverse effects were not statistically significant, with a predictable chronopharmacovigilance illustration.Conclusions: The pharmacotherapeutic efficacy of ofloxacin was more for treating group A, followed by group B, followed by group E and group D, and finally followed by group C. Ofloxacin was safe, without any pharmacogenomic or pharmacogeographic heterogeneity related fluctuation.

Effects of Cullin1 on the Biological Characteristics of Lung Adenocarcinoma A549 and H1395 Cells

背景与目的Cullin1是Cullin家族中有代表性的一员，对细胞周期、转录和信号转导相关蛋白泛素化起重要作用，Cullin1与多种恶性肿瘤的发生发展有着密切的联系。本研究旨在探讨Cullin1对肺腺癌A549和H1395细胞生物功能的影响。方法实时荧光定量聚合酶链式反应（polymerase chain reaction, PCR）检测肺腺癌细胞（A549、H358、H1395、H1650）及人正常肺上皮细胞BEAS-2B中Cullin1 mRNA表达，采用siRNA技术干扰Cullin1 mRNA相对高表达的肺腺癌细胞；采用四甲基偶氮唑盐比色法（methyl thiazolyl tetrazolium assay, MTT）、流式细胞术及Transwell实验检测细胞增殖、细胞周期分布、细胞早期凋亡及侵袭和迁移能力；采用Western blot检测基质金属蛋白酶-2（matrix metalloproteinase-2, MMP-2）、基质金属蛋白酶-9（matrix metalloproteinase-9, MMP-9）、组织基质金属酶抑制剂-1（tissue inhibitor of metalloproteinase-1, TIMP-1）、细胞周期蛋白D1（Cyclin D1）、细胞周期蛋白E2（Cyclin E2）、p21和p27蛋白的表达水平。结果与BEAS-2B细胞相比，肺腺癌细胞中Cullin1 mRNA均呈高表达，尤其在肺腺癌A549和H1395细胞中相对表达量较高（P < 0.05）；干扰Cullin1后肺腺癌细胞的增殖能力受到了抑制，G1期细胞增多，S期细胞数目减少，肺腺癌细胞早期凋亡率明显升高（P < 0.05）；肺腺癌细胞的侵袭及迁移能力下降（P < 0.05）；干扰Cullin1后MMP-9、MMP-2、Cyclin D1及Cyclin E2蛋白表达量减少（P < 0.05），而TIMP-1、p21和p27蛋白表达量增多（P < 0.05）。结论干扰Cullin1后可抑制肺腺癌A549和H1395细胞的增殖、侵袭和迁移，Cullin1在肺腺癌中发挥促癌作用。

Fargesin Inhibits EGF-Induced Cell Transformation and Colon Cancer Cell Growth by Suppression of CDK2/Cyclin E Signaling Pathway.

Although the lignan compound fargesin is a major ingredient in Shin-Yi, the roles of fargesin in carcinogenesis and cancer cell growth have not been elucidated. In this study, we observed that fargesin inhibited cell proliferation and transformation by suppression of epidermal growth factor (EGF)-stimulated G1/S-phase cell cycle transition in premalignant JB6 Cl41 and HaCaT cells. Unexpectedly, we found that signaling pathway analyses showed different regulation patterns in which fargesin inhibited phosphatidylinositol 3-kinase/AKT signaling without an alteration of or increase in mitogen activated protein kinase (MAPK) in JB6 Cl41 and HaCaT cells, while both signaling pathways were abrogated by fargesin treatment in colon cancer cells. We further found that fargesin-induced colony growth inhibition of colon cancer cells was mediated by suppression of the cyclin dependent kinase 2 (CDK2)/cyclin E signaling axis by upregulation of p21WAF1/Cip1, resulting in G1-phase cell cycle accumulation in a dose-dependent manner. Simultaneously, the suppression of CDK2/cyclin E and induction of p21WAF1/Cip1 were correlated with Rb phosphorylation and c-Myc suppression. Taken together, we conclude that fargesin-mediated c-Myc suppression inhibits EGF-induced cell transformation and colon cancer cell colony growth by the suppression of retinoblastoma (Rb)-E2F and CDK/cyclin signaling pathways, which are mainly regulated by MAPK and PKB signaling pathways.

The inhibitory effect of human DEFA5 in growth of gastric cancer by targeting BMI1.

Defensins, a class of small cysteine‐rich cationic polypeptides across cellular life, are identified as antimicrobial compounds that display direct antimicrobial and immune signaling activities that are involved in the host defense. In addition to their roles in the innate immune system, accumulating studies have reported that some members of defensins are expressed and involved in some cancer cells, such as colon cancer, colorectal cancer, lung cancer and renal cell carcinomas. However, the roles of α‐Defensin 5 (DEFA5) in tumorigenesis and development remain unknown. In the present study, bioinformatics analysis and quantitative PCR results showed that the expression level of DEFA5 was dramatically downregulated in human gastric cancer. Overexpression of human DEFA5 in gastric cancer cell lines SGC7901 and BGC823 effectively diminished cell proliferation and reduced the colony forming ability. Moreover, DEFA5 overexpression induced cell cycle arrest by significantly increasing the number of G1‐phase cells. Consistently, in vivo tumor formation experiments in nude mice showed the suppression of the tumor growth by DEFA5 overexpression, suggesting an inhibitory effect of DEFA5 in gastric cancer. Mechanistically, DEFA5 directly binds to BMI1, which subsequently decreased its binding at the CDKN2a locus and upregulated the expression of 2 cyclin‐dependent kinase inhibitors, p16 and p19. Taken together, we concluded that DEFA5 showed an inhibitory effect in gastric cancer cell growth and may serve as a potential tumor suppressor in gastric cancer.

Clinical significance of LARGE1 in progression of liver cancer and the underlying mechanism.

Liver cancer is a malignant disease and causes thousands of death each year. The prognosis is dismal for patients with metastasis and recurrence. It is urgent to disclose the cause and mechanism underlying liver cancer. LARGE1 encodes a glycosyltransferase and was reported to promote progression in cancer. But its role in liver cancer is unknown. In this study, LARGE1 displayed upregulated expression in liver cancer cells. When LARGE1 was knocked down in SMMC-7721 and Huh-7 cells, the ability of cell proliferation and colony formation were decreased significantly. Cell migration and invasion were suppressed. The number of cells in G1 phase increased but decreased in S phase. Cell apoptosis was not affected. Tumor growth in vivo was also inhibited. Tumor volume was decreased from 1270mm3 to 721mm3 (p<0.05) and tumor weight from 0.95g to 0.63g (p<0.05). Furthermore, the expression of β-catenin, TCF and Cyclin D1 was reduced when LARGE1 was knocked down but increased in LARGE1-overexpressed cells. LGK-974, a specific inhibitor in canonical Wnt signaling, inhibited cell proliferation even when LARGE1 was over-expressed. In tumor tissues, LARGE1 was increased by 4.8 folds compared to paratumoral tissues. And higher LARGE1 expression caused shorter survival. Clinicopathological analysis demonstrated that LARGE1 was associated with TNM stage (Ⅰ/Ⅱ vs III/IV, p=0.005). Therefore, LARGE1 promotes progression and regulates Wnt/β-catenin signaling pathway in liver cancer.

Synthesis and biological evaluation of novel withangulatin A derivatives as potential anticancer agents

Novel withangulatin A (WA) derivatives were synthesized and evaluated for antiproliferative activity against four human cancer cell lines (U2OS, MDA-MB-231, HepG2, and A549). Among these derivatives, 10 exhibited the most potent antiproliferative activity, with an IC50 value of 74.0 nM against the human breast cancer cell line MDA-MB-231 and potency that was 70-fold that of WA (IC50 = 5.22 µM). Moreover, 10 caused G2-phase cell cycle arrest in a concentration-dependent manner and induced the apoptosis of MDA-MB-231 cells by increasing intracellular reactive oxygen species (ROS). Compound 10 showed a high selectivity index (SI = 267.03) for breast cancer MDA-MB-231 cells. These results suggest that 10 is a promising anticancer agent.

Effect of miR-214 on Proliferation, Cell Cycle and Apoptosis of Esophageal Cancer Cells

Esophageal cancer seriously affects human health. miR-214 involves in esophageal cancer, but its specific mechanism has not been completely elucidated. Our study investigated miR-214’s role in esophageal cancer. Eca109 cells were transfected with miR-214 inhibitor/NC was transfected into Eca109 cells followed by analysis of miR-214 level by real-time PCR, cell proliferation by CCK8 assay, cell apoptosis and cell cycle and PTEN level by Western blot. miR-214 was significantly upregulated in Eca109 cells (P &lt; 0.01) with downregulated PTEN (P &lt; 0.05). miR-214 inhibitor significantly upregulated PTEN, decreased cell number, increased apoptosis and cells in G1 phase (P &lt; 0.05). PTEN was a target of miR-214. miR-214 affects esophageal cancer cells by targeting PTEN.

673 Oxidative stress promotes pathologic cellular remodeling of the cervix: implications for pregnancy and parturition

A physiologic increase in reactive oxygen species (ROS) throughout pregnancy is required to remodel uterine tissues including the cervix. Oxidative stress (OS) and excessive ROS can cause cellular damages resulting in a dysfunctional tissue. This study determined the effects of OS and OS induced cell fate of cervical epithelial and stromal cells. Cervical cells were isolated and immortalized from biopsies after hysterectomy from women with benign gynecologic conditions. Ectocervical (Ecto) and endocervical epithelial cells (Endo) and cervical stromal cells (CSC) were treated with cigarette smoke extract (CSE; 1:50), an OS inducer, for 48 h. Cell viability (crystal violet assay), cell cycle, apoptosis and necrosis and (flow cytometry), senescence (senescence-associated b-galactosidase staining), autophagy (staining for autophagosome protein LC3B), stress signaler p38MAPK pathway activation (western blot analyses), and sterile inflammation by measuring IL-6 (ELISA) were conducted after 48 h of CSE treatment. Our main findings are as follows: (1) OS induced ROS production in cervical cells that was inhibited by the antioxidant N-acetyl cysteine (NAC). Distinct forms of cell fate induced by OS in different cell types are listed in Table 1. 2) OS induced apoptosis and necrosis in Ecto but only caused necrosis in Endo and CSC. 3) High senescence and low autophagy were observed in CSC under OS. Conversely, senescence was lower and autophagy was higher in Endo; 4) OS decreased cell cycle progressions in G1G0 phase and increased cells in G2 phase indicating a cell cycle arrest; 5) OS induced p38MAPK activation in all three cervical cells (Fig.1A), and increased IL-6 production. Inhibition of IL-6 production by a p38MAPK inhibitor confirmed activation of OS induced stress signaling pathway (Fig.1B). OS can cause cell cycle arrest, promote different forms of cell deaths, and sterile inflammation mediated by p38MAPK activation in the cellular components of the cervix. OS may contribute to accelerating the cervical ripening process which can predispose patients to preterm birth.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Integrative lncRNA–mRNA co‐expression network analysis identifies novel lncRNA E2F3‐IT1 for rheumatoid arthritis

Integrative lncRNA–mRNA co‐expression network analysis identifies novel lncRNA E2F3‐IT1 for rheumatoid arthritis

Bloom syndrome DNA helicase deficiency is associated with oxidative stress and mitochondrial network changes

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.

Regulatory effect and mechanism of APN gene on porcine epidemic diarrhea virus resistance.

The expression level of aminopeptidase N (APN) is evidently correlated with porcine epidemic diarrhea virus (PEDV) infectivity. This study aims to examine the mechanisms regulating APN expression level in response to PEDV infection. Quantitative real time PCR was performed herein to detect gene expression dynamics at various timepoints after PEDV infection. Subsequently, CRISPR/Cas9 gene editing technology was used to generate a APN-knockout IPEC-J2 cell line, exploring the effects of APN on cell proliferation by propidium iodide staining and anti-PEDV activity by indirect immunofluorescence assay. Ultimately, the effects of single nucleotide polymorphisms (SNPs) and methylation in the APN promoter region on gene expression were analyzed by using bisulfite sequencing PCR and dual luciferase reporter gene assay. APN expression was significantly upregulated within 4-24h post-infection. The cytoactivity of the APN-knockout IPEC-J2 cell line was markedly suppressed at different timepoints. Further, cell cycle analyses indicated an increase in the number of G1-phase cells and a significant decrease in that of S-phase cells. Moreover, key cyclical factors regulating the G1 phase were highly expressed in APN-knockout cells. The RNA copies of viral particles and mRNA levels of antiviral genes and inflammatory cytokines in APN-knockout cells were markedly decreased within 24h of PEDV infection. Similarly, indirect immunofluorescence assay confirmed that the number of PEDV particles was significantly decreased. Sequence analysis revealed two CpG islands in the APN promoter region. However, there was no evident correlation between the methylation status of APN promoter and mRNA levels. Dual luciferase reporter gene assay showed that the SNP rs326030589 (G/A) significantly increased the promoter activity of APN. These results suggested that APN knockout enhanced the resistance of IPEC-J2 cells to PEDV. Moreover, rs326030589 in the APN promoter region participated in gene transcription regulation. Our results provide a reference for studying the mechanisms regulating APN and may contribute to the application of APN gene in resistance breeding of swine epidemic diarrhea.

Total Phenolic Fraction (TPF) from Extra Virgin Olive Oil: Induction of apoptotic-like cell death in Leishmania spp. promastigotes and in vivo potential of therapeutic immunomodulation.

Leishmaniasis is a serious multifactorial parasitic disease with limited treatment options. Current chemotherapy is mainly consisted of drugs with serious drawbacks such as toxicity, variable efficacy and resistance. Alternative bioactive phytocompounds may provide a promising source for discovering new anti-leishmanial drugs. Extra Virgin Olive Oil (EVOO), a key-product in the Mediterranean diet, is rich in phenols which are associated with anti-inflammatory, anti-cancer and anti-microbial effects. In this study, we investigate the anti-leishmanial effect of Total Phenolic Fraction (TPF) derived from EVOO in both in vitro and in vivo systems by investigating the contributing mechanism of action. We tested the ability of TPF to cause apoptotic-like programmed cell death in L. infantum and L. major exponential-phase promastigotes by evaluating several apoptotic indices, such as reduction of proliferation rate, sub-G0/G1 phase cell cycle arrest, phosphatidylserine externalization, mitochondrial transmembrane potential disruption and increased ROS production, by using flow cytometry and microscopy techniques. Moreover, we assessed the therapeutic effect of TPF in L. major-infected BALB/c mice by determining skin lesions, parasite burden in popliteal lymph nodes, Leishmania-specific antibodies and biomarkers of tissue site cellular immune response, five weeks post-treatment termination. Our results show that TPF triggers cell-cycle arrest and apoptotic-like changes in Leishmania spp. promastigotes. Moreover, TPF treatment induces significant reduction of parasite burden in draining lymph nodes together with an antibody profile indicative of the polarization of Th1/Th2 immune balance towards the protective Th1-type response, characterized by the presence of IFN-γ-producing CD4+ T-cells and increased Tbx21/GATA-3 gene expression ratio in splenocytes. TPF exhibits chemotherapeutic anti-leishmanial activity by inducing programmed cell death on cell-free promastigotes and immunomodulatory properties that induce in vivo T cell-mediated responses towards the protective Th1 response in experimental cutaneous leishmaniasis. These findings enable deeper understanding of TPF's dual mode of action that encourages further studies.

AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell lymphoma

Angiomotin (AMOT) is a membrane protein that is aberrantly expressed in a variety of solid tumors. Accumulating evidence support that AMOT is involved in the pathological processes of tumor proliferation, apoptosis, and invasion. However, the potential role of AMOT in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains elusive. In the present study, we investigated the expression level and biological function of AMOT in DLBCL. AMOT expression was significantly reduced in DLBCL biopsy section, and low AMOT expression was associated with poor clinical prognosis. Overexpression of AMOT by lentivirus in human DLBCL cells induced cell viability inhibition concomitant with an increased percentage of cells in G1 phase and decreased percentage in S phase. Moreover, AMOT upregulation increased the sensitivity of DLBCL cells to doxorubicin. Furthermore, overexpression of AMOT led to reduced activation of key kinases for the DNA damage response (DDR). The above results indicated that AMOT acts as a tumor suppressor via inhibition of the DDR, thus reducing the viability while increasing the chemosensitivity in DLBCL. In summary, AMOT may be a novel potential target for DLBCL therapeutic intervention.

2,6-DMBQ suppresses cell proliferation and migration via inhibiting mTOR/AKT and p38 MAPK signaling pathways in NSCLC cells

2,6-Dimethoxy-1,4-benzoquinone (2,6-DMBQ) is the major bioactive compound found in fermented wheat germ extract. Although fermented wheat germ extract has been reported to show anti-proliferative and anti-metabolic effects in various cancers, the anticancer potential and molecular mechanisms exerted by 2,6-DMBQ have not been investigated in non-small cell lung cancer (NSCLC) cells. Here, we report that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibiting activation of AKT and p38 MAPK. 2,6-DMBQ significantly suppressed anchorage-dependent and independent cell growth. Additionally, 2,6-DMBQ induced G2 phase cell cycle arrest through inhibiting the expression and phosphorylation of cyclin B1 and CDC2, respectively. Furthermore, 2,6-DMBQ strongly suppressed NSCLC cell migration through induction of E-cadherin expression. To determine the molecular mechanism(s) exerted by 2,6-DMBQ upon NSCLC cell lines, various signaling kinases were screened; the results indicate that 2,6-DMBQ strongly inhibits the phosphorylation of AKT and p38 MAPK. Additionally, the growth kinetics of cells treated with an AKT or p38 MAPK inhibitor in combination with 2,6-DMBQ indicate that 2,6-DMBQ suppresses NSCLC cell growth and migration through inhibition of AKT and p38 MAPK. Taken together, our results suggest that 2,6-DMBQ is a potential anticancer reagent against NSCLC cells and could be useful for treating lung cancer patients.