Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture ...conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.
Highlights • Orphan nuclear receptors (ONRs) were reviewed in the context of prostate and breast cancers. • General structural features and biological functions of ONRs were discussed. • The most ...promising candidates for therapeutic targeting were reviewed. • The availability of small molecules and endogenous ligands were highlighted for each ONR discussed.
Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ...ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.
Human androgen receptor (AR) is a hormone-activated transcription factor that is an important drug target in the treatment of prostate cancer. Current small-molecule AR antagonists, such as ...enzalutamide, compete with androgens that bind to the steroid-binding pocket of the AR ligand-binding domain (LBD). In castration-resistant prostate cancer (CRPC), drug resistance can manifest through AR-LBD mutations that convert AR antagonists into agonists, or by expression of AR variants lacking the LBD. Such treatment resistance underscores the importance of novel ways of targeting the AR. Previously, we reported the development of a series of small molecules that were rationally designed to selectively target the AR DNA-binding domain (DBD) and, hence, to directly interfere with AR-DNA interactions. In the current work, we have confirmed that the lead AR DBD inhibitor indeed directly interacts with the AR-DBD and tested that substance across multiple clinically relevant CRPC cell lines. We have also performed a series of experiments that revealed that genome-wide chromatin binding of AR was dramatically impacted by the lead compound (although with lesser effect on AR variants). Collectively, these observations confirm the novel mechanism of antiandrogen action of the developed AR-DBD inhibitors, establishing proof of principle for targeting DBDs of nuclear receptors in endocrine cancers.
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Introduction: Based on quantitative sensory testing (QST) we described the progression of diabetic sensorimotor polyneuropathy (DSPN) from healthy to thermal hyperalgesia, to mechanical hyperalgesia, ...to sensory loss. While QST provides valuable insights, it gives less information about the level of sensory impairment (transductional vs axonal). Therefore, we performed an electrical stimulation protocol to evaluate C-nociceptor excitability in individuals with DSPN. Methods: We examined 66 individuals with diabetes mellitus, 51 having varying stages of DSPN. Slow depolarizing transcutaneous currents of low-intensity with 4 Hz sinusoidal stimulation profile and single 500 ms half sine wave pulses were used to stimulate C-fibers. Pain perception was quantified using (0-10)-Numeric-Rating-Scale. Findings were cross-referenced with QST Z-scores and correlated to serum levels of the axonal marker neurofilament light chain (NfL). Results: Mechanical pain scores positively correlated with electrically induced pain (r=0.757, P<0.001) whereas only a weak positive correlation was found between electrical pain and thermal pain Z-scores at high stimulation intensities (r=0.403, P<0.01). Increased electrical pain ratings were found in individuals with reduced heat pain but not in participants with reduced mechanical pain indicating a possible axonal degeneration in the latter. In most of our subjective sensory tests, higher NfL levels correlated with impaired sensory function (r>-0.3, P<0.05). Conclusion: Loss of mechanical pain indicated axonal degeneration, while absent heat sensation with normal electrically induced pain suggested a progression involving impaired transduction or superficial degeneration of sensory endings. Hypersensitivity to weak depolarizing stimuli may offer insights into DSPN's natural course, aiding high-risk patient identification and guiding interventions. Disclosure O. Eldesouky: None. L. Seebauer: Other Relationship; Springer Medizin Verlag GmbH. R. Rukwied: None. R. Carr: None. M. Roshan: None. A. Sulaj: None. D. Tsilingiris: None. S. Kopf: Speaker's Bureau; Lilly Diabetes, Bayer Inc. T.H. Fleming: None. M. Schmelz: Consultant; Lilly GmbH, Merz Therapeutics, Bayer Inc., Medtronic GmbH. J. Szendroedi: None. Z. Kender: None.
Introduction & Objective:The description of prediabetes subphenotypes could improve the detection of individuals at increased risk for the onset of diabetes and/or related complications. The aim of ...this study was to assess the prevalence of diabetes-associated complications in newly established clusters of prediabetes and to compare them with the current classification of prediabetes. Methods: People with prediabetes (n=118: 57/61 women/men, HbA1c 5.6±0.3%, age 57.4±9.8 y, BMI 29.7±5.7 kg/m2) were identified either using classical clinical parameters (increased HbA1c levels: HbA1c, impaired fasting glucose: IFG, impaired glucose tolerance: IGT) or using a novel pathophysiology-based subphenotyping of prediabetes (cluster 1-6, according to Wagner et al., Nature medicine 2021 ). Study participants were screened for diabetes-associated complications such as nephropathy, neuropathy, retinopathy, and pneumopathy, with a focus on liver steatosis and fibrosis according to liver stiffness measurement. Results: Individuals allocated to the new prediabetes clusters 5 and 6 showed more often at least 2 diabetes-associated complications (70.6% vs. 52.2%) compared to prediabetes individuals identified using the classical parameters HbA1c, IFG and IGT (44.3% vs. 37.3% vs. 16.7%), all p=0.026. Elevated markers for liver steatosis were more common in cluster 3, 4, 5 and 6 (76% vs. 91% vs. 100% vs. 83%) compared to classical cluster HbA1c (74%), whereas for liver fibrosis in cluster 5 and 6 (50% vs. 41%) compared to 26% in the IFG cluster. Further analyses showed that 83% and 73% of people in cluster 5 and 6 with liver steatosis had at least one additional diabetes-associated complication compared to 63%/56% in cluster HbA1c/IFG. Conclusion: The novel pathophysiology-based clusters of prediabetes help to better identify people with increased risk of diabetes-associated complications, allowing the design of early targeted preventive measures. Disclosure M. Ansmann: None. E. Kliemank: None. E.V. Rauchhaupt: None. L. Schimpfle: None. L. Seebauer: Other Relationship; Springer Medizin Verlag GmbH. M. Roshan: None. Z. Kender: None. S. Kopf: Speaker's Bureau; Lilly Diabetes, Bayer Inc. J. Szendroedi: None. A. Sulaj: None.
Introduction & Objective: We aimed to assess C-fiber excitability via transcutaneous-electrical nerve stimulation (TENS) in diabetic sensorimotor neuropathy (DSPN) and its association with epidermal ...innervation and quantitative sensory testing. Pain perception was evaluated in glucose-tolerant humans (n=14) and patients with type 2 diabetes: no-DSPN (n=15), possible-DSPN (n=21), probable-DSPN (n=19), and confirmed-DSPN (n=11). Subsequent analysis compared patients with positive (n=17) and negative symptoms (n=16). Methods: Slow depolarizing transcutaneous currents of low-intensity with 4 Hz sinusoidal stimulation profile and single 500 ms half sine wave pulses were used to stimulate C-fibers. Pain perception quantified using (0-10)-Numeric-Rating-Scale, detection/pain thresholds and pain-change-dynamics were assessed. Results: Confirmed-DSPN had diminished pain perception (1.72±0.50 vs 4.98±0.46), elevated detection (0.19±0.04 vs 0.05±0.01) and pain thresholds (0.28±0.04 vs 0.1±0.01) and reduced pain habituation compared to the control group. Elevated pain perception (pos:7.32±0.72 vs neg:3.81±0.98) and lowered detection/pain thresholds (pos:0.24±0.04 vs neg:0.13±0.03) were shown in the positive symptoms group compared to the negative symptoms group (p<0.05). Over 50% of patients with DSPN experience elevated pain with electrical stimuli in regions with reduced heat pain sensitivity, while less than 6% show similar responses in areas with diminished mechanical pain sensitivity indicating axonal degeneration. Conclusions: Our results suggest that DSPN proceeds via differential transductional disruption towards denervation. TENS results, influenced by positive symptoms, confirm C-fibers' role for the clinical burden of DSPN. TENS offers further insights into DSPN's natural course, aiding high-risk patient identification and guiding interventions. Disclosure O. Eldesouky: None. L. Seebauer: Other Relationship; Springer Medizin Verlag GmbH. R. Rukwied: None. R. Carr: None. M. Roshan: None. A. Sulaj: None. D. Tsilingiris: None. S. Kopf: Speaker's Bureau; Lilly Diabetes, Bayer Inc. T.H. Fleming: None. M. Schmelz: Consultant; Lilly GmbH, Merz Therapeutics, Bayer Inc., Medtronic GmbH. J. Szendroedi: None. Z. Kender: None.
Abstract only Objective: Neuregulin-1 (Nrg1) promotes cardiomyocyte hypertrophy, survival, and cell cycle activity through ErbB signaling. It is investigated in clinical trials as a cardioprotective ...agent; however, its therapeutic use might be limited due to its pro-neoplastic potential for non-cardiomyocytes that makes targeted approaches necessary. High-throughput screening of hypertrophic agonists found that Nrg1 induces CITED4 (C4) expression. C4 is also upregulated in both physiological and pathological cardiac growth and necessary to prevent adverse remodeling in vivo. However, how C4 regulates Nrg1-signaling in the heart is yet unknown. Methods: We combined pulsed-SILAC labeling, click-chemistry and mass spectrometry that allows to capture immediate changes in the proteome and secretome after Nrg1 (12h and 24h) stimulation in siRNA-mediated C4-knockdown (C4KD) and control (ctr) NRVM and complemented this with established cell- and molecular biology techniques to validate and investigate cellular function and molecular signaling. Results: We confirmed dose-dependent C4 mRNA upregulation in response to Nrg1 stimulation in NRVM. Nrg1 downstream signaling through AKT was hindered in C4KD. Computational analysis comparing the nascent proteome after Nrg1 stimulation in C4KD and ctr NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo , we found significant upregulation of TGFb2 in C4KD NRVM. We next confirmed the upregulation of TGFb2 and its downstream effectors in C4KD and ctr, while adenoviral overexpression of C4 led to reduced TGFb2 expression. Additionally, we found TGFb2 secretion increased from C4KD NRVM with Nrg1 stimulation in the nascent secretome. Conditioned media from C4KD NRVM led to an increased pro-fibrotic response in cardiac fibroblasts. In conclusion, nascent proteomics and secretomics help to elucidate C4-dependent Nrg1-signaling, which may be an important contribution toward our goal to identify targetable cardioprotective pathways in the heart.
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