Epac1 is involved in cell cycle progression in lung cancer through PKC and Cx43 regulation

Introduction. The exchange protein directly activated by cAMP (Epac1), a downstream target of the second messenger cAMP, modulates multiple biological effects of cAMP, alone or in cooperation with protein kinase A (PKC). Epac1 is necessary for promoting protein kinase C (PKC) translocation and activation. The aim of the study was to assess the intensity of Epac1 and protein kinase C (PKC) immunoreactivity in lung cancer and para-carcinoma tissues, and their associations with clinical-pathological indexes. Correlations between the immunoreactivity of Epac1, PKC, A-kinase anchor protein 95 (AKAP95) and connexin43 (Cx43) were also examined. Material and methods. Epac1, Cx43 (46 cases) and PKC, AKAP95 (45 cases) immunoexpression levels were determined in tissue samples of lung cancer and in 12 samples of neighboring para-carcinoma specimens by the PV-9000 Two-step immunohistochemical technique. Results. The percentage of Epac1 positive samples was significantly lower in lung cancer tissue than in neighboring para-carcinoma specimens (37% vs. 83.3%, p < 0.05); the difference in PKC immunoreactivity was not significant (64.4% vs. 91.7%). Epac1 expression was associated with the degree of malignancy and lymph node metastasis (P < 0.05), but not with histological type (P > 0.05), whereas PKC expression was not related to these parameters. Interestingly, Epac1 expression was correlated with PKC and Cx43 expression. Moreover, PKC expression was correlated with AKAP95 expression. Conclusion. Normal Epac1 expression may suppress lung cancer occurrence and metastasis, and its downregulation is involved in cell cycle progression in lung cancer through PKC and Cx43 regulation.

Low follistatin level is a causal risk factor for spontaneous abortion: a two-sample mendelian randomization study

BackgroundRecurrent pregnancy loss is a distressing event during pregnancy, and understanding its causal factors is crucial. Follistatin, a glycoprotein involved in folliculogenesis and embryogenesis, has been implicated as a potential contributor to the risk of spontaneous abortion. However, establishing a causal relationship requires rigorous investigation using robust methods.MethodsIn this study, we utilized mendelian randomization (MR), a powerful genetic epidemiological approach, to examine the causal relationship between follistatin levels and spontaneous abortion. We obtained instrumental variables strongly associated with follistatin levels from large-scale genome-wide association from the IEU database. The inverse variance weighting (IVW) method was taken as gold standard. We also performed sensitivity test to evaluate the robustness of our result.ResultsMR analysis revealed a significant causal relationship between low follistatin levels and spontaneous abortion (p = 0.03). Sensitivity analyses, including pleiotropy test, heterogeneity test, and leave-one-out analysis, all supported the robustness of our findings.ConclusionOur study provides compelling evidence supporting the causal relationship between low follistatin levels and increased risk of spontaneous abortion. These findings underscore the importance of follistatin in the etiology of spontaneous abortion and suggest potential preventive interventions. Modulating follistatin levels or relevant pathways could hold promise for reducing the incidence of spontaneous abortion and improving reproductive outcomes. The utilization of MRs strengthens the validity of our results by mitigating confounding and reverse causality biases. Further research is needed to elucidate the underlying molecular mechanisms and explore therapeutic strategies targeting follistatin levels

RNAi-Mediated c-Rel Silencing Leads to Apoptosis of B Cell Tumor Cells and Suppresses Antigenic Immune Response In Vivo

c-Rel is a member of the Rel/NF-κB transcription factor family and is predominantly expressed in lymphoid and myeloid cells, playing a critical role in lymphocyte proliferation and survival. Persistent activation of the c-Rel signal transduction pathway is associated with allergies, inflammation, autoimmune diseases, and a variety of human malignancies. To explore the potential of targeting c-Rel as a therapeutic agent for these disorders, we designed a small interfering RNA (siRNA) to silence c-Rel expression in vitro and in vivo. C-Rel-siRNA expression via a retroviral vector in a B cell tumor cell line leads to growth arrest and apoptosis of the tumor cells. Silencing c-Rel in primary B cells in vitro compromises their proliferative and survival response to CD40 activation signals, similar to the impaired response of c-Rel knockout B cells. Most important, in vivo silencing of c-Rel results in significant impairment in T cell-mediated immune responses to antigenic stimulation. Our study thus validates the efficacy of c-Rel-siRNA, and suggests the development of siRNA-based therapy, as well as small molecular inhibitors for the treatment of B cell tumors as well as autoimmune diseases

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Discovery of a Selective Binder of Proteasomal Subunit RPN-6 and its Effect on Proteasome Activity

The ubiquitin-proteasome system is responsible for cellular protein recycling, and it is a crucial system to maintain proper protein balances in cells. Proteasome is the main component of the system, and the system is tightly related to multiple cellular processes. Malfunction of the proteasome could lead to various diseases including cancer, neurodegenerative diseases and autoimmune diseases. As a result, researchers have been developing small molecules to target the proteasome to regulate its function. Currently, three small molecules have been approved by FDA as proteasome inhibitors to treat hematological cancer multiple myeloma. However, these small molecules inhibit the same enzymatic subunit on the proteasome and drug resistance has been observed among patients administrating these proteasome inhibitors. To develop new small molecules to target the proteasome, we started to investigate the 19S regulatory particle of the proteasome. In this work, we presented a workflow of discovering a small molecule selective binder, TXS-8, to 19S regulatory particle subunit Rpn-6. We also developed a series of assays to investigate the impact of small molecule on proteasome activity. At last, we introduced the binding site study of TXS-8, development of TXS-8-based PROTAC and new proteasome probe development. We first developed a one-bead-one-compound (OBOC) library to screen with Rpn-6 to discover potential binders to Rpn-6. After careful evaluation and validation, TXS-8 was discovered as the best hit from the screening. Our covalent pull-down experiment with cell lysate later confirmed TXS-8 as a selective binder of Rpn-6 and proteomic analysis of the pulled down protein also validated Rpn-6 as the major target of TXS-8. We then investigated the impact of TXS-8 in Rpn-6 overexpressed cancer cells like Ramos B-cell and multiple myeloma. TXS-8 was four-fold more toxic in these cells comparing to our control HEK-293T cells. To understand the cause of cell death when dosed with TXS-8, we began to investigate the impact of TXS-8 on proteasome activity, but some preliminary results were inconsistent. By the same time, there is also lack of a general workflow to investigate the impact of small molecules on proteasome activity. Therefore, we developed a three-step process to illustrate the general workflow using TXS-8 as an example. We first knocked down Rpn-6 in HEK293T cells and monitored proteasome activity changes with a cell permeable probe our lab developed. We then transfected HEK-293T cells with a full-length foreign protein and knocked down Rpn-6 in these cells. We later monitored the degradation of the foreign protein when dosed with TXS-8. In the last step, we monitored the proteasome activity changes in primary cell lines when dosed with TXS-8. From these three steps, we successfully demonstrated a general workflow to investigate if a small molecule can affect proteasome activity. We also concluded that TXS-8 was unable to affect proteasome activity at non-lethal concentration. To further investigate TXS-8 and provide guidance for future structural optimization to improve potency, we proposed two methods on investigating the general binding site of TXS-8 on Rpn-6 using cross-linking techniques that is currently ongoing. We also modified TXS-8 into proteolysis targeting chimeras (PROTACs) to investigate if TXS-8-based PROTAC can improve toxicity and selectively induce Rpn-6 degradation in cells. However, no significant cell toxicity or Rpn-6 degradation was observed when dosed with TXS-8-based PROTACs. Finally, Due to limitation of cell permeable probes, we were unable to investigate the impact of TXS-8 on the caspase-like β1 and trypsin-like β2 subunit of the proteasome in our previous studies. Although TXS-8 did not alter the chymotrypsin-like activity at non-lethal concentration, examining the effect of TXS-8 on the caspase-like and trypsin-like activity could still benefit our research. Besides, we also desire to expand our proteasome activity toolbox by developing more sensitive proteasome probes. Therefore, by analyzing and combing the commercially available proteasome probes and LLVY-Rh probes, we decided to develop selective proteasome probes for the β1 and β2 subunit to provide useful tools for future potential small molecule proteasome regulator characterization

Discovery of a Small Molecule Probe to Rpn-6, an Essential Subunit of the 26S Proteasome

Rpn-6 is among several essential proteins that facilitate assembly of the 26S proteasome. We were interested in discovering a small molecule binder to Rpn-6 that could be used to further our understanding of the association of the 19S regulatory particle with the 20S core particle and if a small molecule-Rpn-6 interaction could potentially be cytotoxic to cancer cells that heavily rely on proteasome activity for survival. A workflow to utilize a one-bead-one-compound library and a thermal shift assay was developed to discover such a molecule. TXS-8 was discovered to have low micromolar range binding affinity for Rpn-6 and showed with very limited binding to other proteins. Cytotoxicity of TXS-8 was evaluated in several cell lines, revealing increased cytotoxicity to hematological cancers

In vitro silencing of c-Rel led to impaired cell survival and cell cycle progression in primary B cells.

<p>(a) Primary B cells isolated from mouse spleen were stimulated for 24 hr with anti-CD40 (10 µg/ml) before addition of the retroviruses. Cells were harvested at 48 hr post-infection and were monitored by flow cytometry for the infection efficiency. (b) Cell survival and cell cycle progression was analyzed by PI staining. (c) Cells harvested from the same culture in (a) were stained with anti-Ki-67 using intracellular staining methods and analyzed by flow cytometry. The number represents the percentage of Ki-67 positive cells within the GFP⁺ population.</p