Role of Combination Treatment of Aspirin and Zinc in DMH-DSS Induced Colon Inflammation, Oxidative Stress and Tumor Progression in Male BALB/C Mice

Colitis-associated colorectal cancer serves as a prototype of inflammation-associated cancers which is linked with repeated cycles of inflammation and DNA repair deficits. Several preclinical and clinical data reported that aspirin has chemo preventive effect in colorectal cancer and is associated with dose dependent side effects. Further, it has been reported that zinc supplementation improves the quality of life in patients undergoing chemotherapy by alteration of colonic cancer cell gene expression. However, explication of the detailed molecular mechanisms involved in combined administration of aspirin and zinc mediated protection against the colitis associated colorectal cancer deserves further investigation. For the induction of colitis associated colorectal cancer, male BALB/c mice were administered 1, 2-dimethylhydrazine dihydrochloride (DMH) 20 mg/kg/bw thrice, before the initiation of every DSS cycle (3%w/v in drinking water). One week after the initiation of DSS treatment, aspirin (40 mg/kg; p.o.) and zinc in the form of zinc sulphate (3 mg/kg; p.o.) was administered for 8 weeks. Combination of aspirin and zinc as intervention significantly ameliorated DAI score, myeloperoxidase activity, histological score, apoptotic cells and protein expression of various inflammatory markers including nuclear factor kappa light chain enhancer of activated B cells (NFκBp65), cycloxygenase -2 (COX-2), interleukin-6 (IL-6); proliferation markers such as proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription 3 (STAT3) expression significantly decreased and antioxidant enzymes nuclear factor erythroid 2–related factor 2 (Nrf-2), metallothionein, catalase and superoxide dismutase (SOD) significantly increased as evaluated by immunohistochemistry and western blot analysis.

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma

Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

Proliferative activity of the epithelium in squamous epithelial lesions of the cervix

As a criterion for precancerous changes in the stratified squamous epithelium of the cervix, its proliferative activity, studied using monoclonal antibodies PC-10 to the antigen of proliferating cell nuclei (PCNA), is considered. The results of the studies showed that patients with a low degree of squamous intraepithelial lesion are characterized by weak proliferative activity, and for patients with a high degree of lesion it is moderate and pronounced. An increase in proliferative activity is a prognostic factor that determines long-term persistence and the likely progression of the lesion.

Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

ABSTRACTSARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase of PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Corneal Epithelial Removal with a Newly Designed Epithelial Brush

This study aimed to evaluate and compare the effectiveness of a newly developed epithelial removal brush with conventional methods in a rabbit model of corneal epithelial defects. The corneal epithelia of thirty-seven rabbits were removed by three different methods including blades (blade group), newly developed epithelial brushes (Ocu group), and conventional rotating brushes (Amo group). The defect area was measured with light microscopy immediately and at 4, 18, 24, and 50 hours after removal. Corneas were obtained immediately and at 24 and 50 hours and subjected to hematoxylin and eosin (H&E) and immunofluorescence staining using proliferating cell nuclear antigen (PCNA) and phosphorylated heat shock protein 27 (pHSP27) antibodies. The residual stromal surface was observed by scanning electron microscopy (SEM). In the Ocu group, epithelia were significantly recovered at 18, 24, and 50 hours compared with immediately after removal, and in the blade and Amo groups, epithelia were significantly recovered only at 50 hours after epithelial removal. The expression levels of PCNA and pHSP27 did not differ among three groups. There was significantly more inflammatory cell infiltration in the blade group than in the other groups. SEM showed a more regular and uniform residual stromal surface in the Ocu group than in the other groups. The newly developed epithelial brush showed better polishing ability and led to earlier significant epithelial recovery and a more regular and uniform stromal surface than conventional methods in this rabbit model of epithelial defects. Accumulation of clinical data is expected to expand the scope of application of new brushes for laser surface ablation.

The legume-specific transcription factor E1 controls leaf morphology in soybean

Background The leaf is a determinate organ essential for photosynthesis, whose size and shape determine plant architecture and strongly affect agronomic traits. In soybean, the molecular mechanism of leaf development is not well understood. The flowering repressor gene E1, which encodes a legume-specific B3-like protein, is known to be the gene with the largest influence on soybean flowering and maturity. However, knowledge of its potential other functions remains poor. Results Here, we identified a novel function of E1 protein in leaf development. Unifoliolate leaves of E1-overexpression (E1-OE) lines were smaller and curlier than those of wild type DongNong 50 (DN50) and Williams 82 (W82). Transverse histological sections showed disorganized cells and significantly elevated palisade tissue number, spongy tissue number, and bulliform cell number in E1-OE lines. Our results indicate that E1 binds to the promoters of the leaf- development-related CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor genes to negatively regulate their expression. Conclusions Our findings identify E1 as an important new factor in soybean leaf development.

Synthesis and Antiproliferative Activity of 2,4,6,7-Tetrasubstituted-2H-pyrazolo[4,3-c]pyridines

A library of 2,4,6,7-tetrasubstituted-2H-pyrazolo[4,3-c]pyridines was prepared from easily accessible 1-phenyl-3-(2-phenylethynyl)-1H-pyrazole-4-carbaldehyde via an iodine-mediated electrophilic cyclization of intermediate 4-(azidomethyl)-1-phenyl-3-(phenylethynyl)-1H-pyrazoles to 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridines followed by Suzuki cross-couplings with various boronic acids and alkylation reactions. The compounds were evaluated for their antiproliferative activity against K562, MV4-11, and MCF-7 cancer cell lines. The most potent compounds displayed low micromolar GI50 values. 4-(2,6-Diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol proved to be the most active, induced poly(ADP-ribose) polymerase 1 (PARP-1) cleavage, activated the initiator enzyme of apoptotic cascade caspase 9, induced a fragmentation of microtubule-associated protein 1-light chain 3 (LC3), and reduced the expression levels of proliferating cell nuclear antigen (PCNA). The obtained results suggest a complex action of 4-(2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol that combines antiproliferative effects with the induction of cell death. Moreover, investigations of the fluorescence properties of the final compounds revealed 7-(4-methoxyphenyl)-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine as the most potent pH indicator that enables both fluorescence intensity-based and ratiometric pH sensing.

Identification of a Novel Proliferating Cell Fraction in Chronic Lymphocytic Leukaemia with High Expression of IgM and Chemokine Receptors

Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant CD5+ B cells in the peripheral blood (PB), secondary lymphoid tissues and bone marrow. Currently considered an incurable disease, B cell receptor (BCR) signalling plays a key role in the disease aetiology as evidenced by the therapeutic success of BCR signalling inhibitors such as ibrutinib. Previous studies using incorporation of 2H-labelling of DNA in vivo demonstrated sub-clonal heterogeneity in PB CLL cell fractions sorted based on reciprocal densities of chemokine C-X-C motif receptor 4 (CXCR4) and CD5. The CXCR4 loCD5 hi fraction was shown to be enriched in recently born proliferating cells while the CXCR4 hiCD5 lo fraction consists of resting, quiescent cells thought to reflect their migratory and BCR signalling histories in tissue. Whilst these proliferating/resting fractions have since been more closely examined, the remaining bulk PB CLL population has been left relatively unexplored leaving other therapeutically relevant cell fractions undetected. Here, we have comprehensively analysed the phenotype of subpopulations of PB cells from 11 CLL patients using flow cytometry to identify activated and proliferating cell fractions. CD19 +CD5 +cells were divided into 9 fractions based on CXCR4/CD5 densities and to permit comparisons between fractions, each cell fraction was defined as containing 1-2% of the total clonal CD19 +CD5 + population. Surprisingly, we detected enrichment for Ki67+ proliferating cells and high expression of AID in the cell fraction with highest expression levels of both CXCR4 and CD5 (CXCR4 hiCD5 hi), demonstrating that CXCR4 loCD5 hi cells are not the only proliferating fraction in the blood. Moreover, we could detect mitotic cells in the CXCR4 hiCD5 hi fraction using imaging flow cytometry of a nuclear stain. This CXCR4 hiCD5 hi fraction showed the highest surface expression levels of IgM, CD86, CCR7, CXCR3 and CXCR5 of all the fractions assessed (p<0.05), indicating they are highly activated and primed for migration to lymph nodes (LNs) for further activation and proliferation. Proliferation of CLL cells is highest in secondary lymphoid tissues, however the phenotype of proliferating cells in tissue is unknown. To examine the phenotype of proliferating CLL cells in LNs, we analysed a fine-needle aspirate obtained from an enlarged cervical node using flow cytometry and compared this to a matched PB sample. Flow cytometric gates set on the PB sample were used to define and quantify LN cell fractions. Expression levels of both Ki67 and surface IgM were highest in the CXCR4 hiCD5 hi fraction which was expanded to 20% of the CD19 +CD5 + population in the LN whilst CXCR4 loCD5 hi cells (accounting for 2% of the bulk LN population) expressed very low surface IgM and Ki67 levels, suggesting CXCR4 hiCD5 hi cells may be the most proliferative cells in CLL. The CXCR4 loCD5 hi cell fraction has been shown to be a key target of ibrutinib, however the impact of ibrutinib on the CXCR4 hiCD5 hi fraction is unknown. Administration of ibrutinib to PB CLL cells for 48hr in vitro resulted in selective targeted depletion of the CXCR4 loCD5 hi fraction, as evidenced by induction of apoptotic markers in this compartment; conversely, persistent cells after 48hr ibrutinib administration in vitro were exclusively of the CXCR4 hi phenotype. In conclusion, we have identified a potentially dangerous fraction of proliferating cells in the PB of CLL patients with high expression of CXCR4, CD5, IgM, CCR7, CXCR3 and CXCR5 open for both migration to tissue and reception of BCR signals. Furthermore, CXCR4 hiCD5 hi cells in the periphery may closely mirror tissue-resident activated cell phenotypes and may represent critical targets for therapeutic intervention, particularly in high-risk CLL patients refractory to BCR inhibitor therapies. Disclosures Patten: ROCHE: Research Funding; GILEAD SCIENCES: Honoraria, Research Funding; NOVARTIS: Honoraria; JANSSEN: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.

The antioxidant effect of Michauxia campanuloides on rat ovaries

This study investigated how Michauxia campanuloides affects the Proliferating Cell Nuclear Antigen (PCNA) expression in granulosa cells, the ovarian histomorphology and serum total antioxidant capacity (TAC) in rats. Rats were divided into control (C), treatment 1 (T 1) and treatment 2 (T 2) groups. The rats in the T 1 and T 2 groups received aqueous extract of M. campanuloides at doses of 20 mg/kg/day and 40 mg/kg/day orally for 21 days, respectively. Serum TAC levels, follicles counts including primordial, primary, preantral, antral and atretic follicles, and PCNA expression in granulosa cells were evaluated. Numbers of preantral follicles increased in T 1 and T 2 groups compared to C group (P < 0.05). TAC and numbers of preantral and antral follicles increased in T 2 group compared to T 1 and C groups (P < 0.05). PCNA expression in granulosa cells was increased in T 2 group compared to T 1 and C groups (P < 0.01). In conclusion, treatment with M. campanuloides had positive effects on antioxidant activity, follicular dynamics, and PCNA expression of granulosa cell in rats.