G1 Phase Research Articles

Synthesis and anti-cancer investigations of copper(II) complexes based on adenine

Platinum-based chemotherapeutics have played a critical role in oncology for decades. However, their broader utility is hindered by the advent of severe side effects and the emergence of drug resistance. The pursuit of alternative agents, particularly non-platinum (non-Pt) metal complexes, has gained momentum in current research. Designing efficacious non-Pt metal agents that target DNA poses a complex challenge. In this study, we present the strategic design, synthesis, and thorough characterization of two innovative copper(II) complexes leveraging adenine as a ligand, a potential avenue to overcome these challenges. Our investigation demonstrates the superior cytotoxicity of these copper(II) complexes compared to the benchmark cisplatin, with complex C2 exhibiting the most promising anticancer activity, showcasing an impressive IC50 value of 4.51 μM in MGC-803 cells. Mechanistic insights underscore that complex C2 executes its cytotoxic effects by instigating DNA damage, orchestrating cell cycle arrest at the G2 phase, perturbing mitochondrial membrane potential, inducing ROS production, and ultimately triggering apoptotic pathways. These findings significantly emphasize the potential of designing novel adenine-based anticancer metal complexes targeting DNA, portraying a compelling trajectory for advancing anticancer drug development.

Unveiling a cuproptosis-related risk model and the role of FARSB in hepatocellular carcinoma

BackgroundCuproptosis, a type of regulated cell death that was recently identified, has been linked to the development of a variety of diseases, among them being cancers. Nevertheless, the prognostic significance and therapeutic implications of the cuproptosis potential index in hepatocellular carcinoma (HCC) remain uncertain. MethodsSingle-sample gene set enrichment analysis (ssGSEA) and Weighted Gene Co-expression Network Analysis (WGCNA) methodology was conducted to ascertain the identification of modular genes that are closely linked to cuproptosis. In addition, the gene signature indicative of prognosis was formulated by employing univariate Cox regression analysis in conjunction with a random forest algorithm. The efficacy of this gene signature in predicting outcomes was confirmed through validation in both The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets. Furthermore, a study was undertaken to evaluate the association between the risk score and various clinical-pathological characteristics, explore the biological processes linked to the gene signature, and analyze tumor mutational burden and somatic mutations. Lastly, potential drugs targeting the identified gene signature were identified through screening. ResultsThe results of our comprehensive analysis across multiple cancer types demonstrated a positive correlation between an elevated cuproptosis potential index (CPI) and an accelerated rate of tumor progression. Furthermore, employing the WGCNA technique, we successfully identified 640 genes associated with cuproptosis. Among these genes, we meticulously screened and validated a seven-gene signature (TCOF1, NOP58, TMEM69, FARSB, DHX37, SLC16A3, and CBX2) that exhibited substantial prognostic significance. Using the median risk score, the division of HCC patients into cohorts with high- and low-risk highlighted significant disparities in survival results, wherein the group with higher risk exhibited a less favorable overall survival. The risk score exhibited commendable predictive efficacy. Moreover, the in vitro knockdown of FARSB significantly hindered cell viability, induced G1 phase arrest, increased apoptosis, and impaired migration in HepG2 and Huh7 cells. ConclusionOur research has successfully identified a strong seven-gene signature linked to cuproptosis, which could be utilized for prognostic evaluation and risk stratification in patients with HCC. Furthermore, the discovered gene signature, coupled with the functional analysis of FARSB, presents promising prospects as potential targets for therapeutic interventions in HCC.

Circular RNA hsa_circ_0005939 Regulates UHRF1BP1L Expression by Targeting miR-4693-3p to Promote Colorectal Cancer Progression.

Colorectal cancer (CRC) is the second most common and fatal cancer in China. circRNAs are different expressed between tumor and non-tumor tissues, and they are proved to be correlated with tumorigenesis and cancer progression. We aimed to explore the biological and molecular function of hsa_circ_0005939 in CRC. We collected and compared ten CRC tissues and four noncancerous tissues and performed circRNA sequencing. We investigated the hsa_circ_0005939 expression in fresh tissues from CRC and adjacent tissues by qPCR. Meanwhile, functional roles of hsa_circ_0005939 in CRC cells were explored by CCK-8, colony formation, wounding healing, cell apoptosis and western blot assays. RNA-FISH was used to confirm the cellular distribution of hsa_circ_0005939. Bioinformatic prediction and luciferase reporter assay were used to determine the mechanisms of hsa_circ_0005939. Our results indicated that hsa_circ_0005939 was up-regulated in CRC tissues and cells. Up-regulation of hsa_circ_0005939 was associated with the occurrence and the number of lymph node metastasis of CRC. Hsa_circ_0005939 down-regulation inhibited cell proliferation, increased cell apoptosis and caused G2 phase arrest of CRC cells. Mechanistically, luciferase assay revealed that hsa_circ_0005939 acts as a molecular sponge for miR-4693-3p and then enhanced Ubiquitin Like With PHD And Ring Finger Domains 1 binding protein 1 like (UHRF1BP1L) expression. Our findings indicated an oncogenic role of hsa_circ_0005939 in CRC, and it enhanced malignant phenotypes of CRC cells through miR-4693-3p/UHRF1BP1L axis. Our study may offer promising biomarkers and therapeutic targets for CRC.

KNTC1 knockdown inhibits the proliferation and migration of osteosarcoma cells by MCM2.

Osteosarcoma (OS) is a common primary malignant bone tumor, and it is necessary to further investigate the molecular mechanism of OS progression. The expression of kinetochore associated protein 1 (KNTC1) and minichromosome maintenance 2 (MCM2) was detected by immunohistochemistry, quantitative PCR (qPCR) and Western blot. Gene knockdown or overexpression cell models were constructed and the proliferation, apoptosis, cell cycle and migration were detected in vitro, besides, xenograft models were established to explore the effects of KNTC1 downregulation in vivo. Public databased and bioinformatics analysis were performed to screen the downstream molecules and determine the expression of MCM2 in cancers. KNTC1 was overexpressed in OS tissues and positively correlated with overall survival of OS patients. KNTC1 knockdown inhibited the proliferation and migration, and arrested G2 phase, and induced apoptosis. Besides, KNTC1 downregulation restricted the xenograft tumor formation. MCM2, one of the coexpressed genes, was highly expressed in sarcoma and downregulated after KNTC1 knockdown. MCM2 overexpression heightened the proliferation and migration ability of OS cells, which was reversed the inhibiting effects of KNTC1 knockdown. KNTC1 was overexpressed in OS and promoted the progression of OS by upregulating MCM2.

2-Aryl-1H-imidazo[4,5-f][1,10]phenanthroline-Based Binuclear Ru(II)/Ir(III)/Re(I) Complexes as Mitochondria Targeting Cancer Stem Cell Therapeutic Agents.

A series of novel Ru(II)/Ir(III)/Re(I)-based organometallic complexes [Ru2L1, Ru2L2, Ir2L1, Ir2L2, Re2L1, and Re2L2] have been synthesized to assess their potency and selectivity against multiple cancer cells A549, HCT-116, and HCT-116 colon CSCs. The cytotoxic screening of the synthesized complexes has revealed that complex Ru2L1 and Ir2L2 are two proficient complexes among all, but Ru2L1 is the most potent complex. A significant binding constant value was observed for DNA and BSA in all complexes. Significant lipophilic properties allow them to penetrate cancer cell membranes, and substantial quantum yield (ϕf) values support bioimaging potential. Again, these complexes are particular for mitochondrial localization and produce a profuse amount of ROS to damage the mitochondrial DNA and then G1 phase cell-cycle arrest. Protein expression analysis unveiled that pro-apoptotic Bax protein overexpressed in Ru2L1-treated cells, whereas antiapoptotic Bcl-2 protein was expressed twofold in Ir2L2-treated cells, which correlated with autophagy reticence.

Bone marrow mesenchymal stem cells-derived exosomal lncRNA GAS5 mitigates heart failure by inhibiting UL3/Hippo pathway-mediated ferroptosis

BackgroundExosomes (Exos) are involved in the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) on heart failure (HF). We investigated the molecular mechanisms underlying the involvement of BMSC-Exos in ferroptosis on HF.MethodsA rat model of HF and cellular model of hypoxia were established. BMSC-Exos were injected into model rats or co-cultured with model cells. In model rats, the cardiac function (echocardiography), oxidative stress (commercial kits), pathological damage (HE staining), fibrosis (MASSON staining), iron deposition (Prussian blue staining), and cell apoptosis (TUNEL staining) were examined. Viability (cell counting kit-8; CCK-8), cell cycle (flow cytometry), oxidative stress, and Fe2+ levels were detected in the model cells. GAS5, UL3, YAP, and TAZ expression were detected using qRT-PCR, western blotting, and immunohistochemistry analyses.ResultsBMSC-Exos restored cardiac function and inhibited oxidative stress, apoptosis, pathological damage, fibrosis, and iron deposition in myocardial tissues of HF rats. In hypoxic cells, BMSC-Exos increased cell viability, decreased the number of G1 phase cells, decreased Fe2+ levels, and inhibited oxidative stress. Ferrostatin-1 (a ferroptosis inhibitor) exhibited a synergistic effect with BMSC-Exos. Additionally, GAS5 was upregulated in BMSC-Exos, further upregulating its target UL3 and Hippo pathway effectors (YAP and TAZ). The relieving effects of BMSC-Exos on HF or hypoxia-induced injury were enhanced by GAS5 overexpression, but weakened by UL3 silencing or verteporfin (a YAP inhibitor).ConclusionsGAS5-harbouring BMSC-Exos inhibited ferroptosis by regulating the UL3/Hippo pathway, contributing to HF remission in vivo and in vitro.

Design, semi-synthesis and bioevaluation of koumine-like derivatives as potential antitumor agents in vitro and in vivo.

Aims: Five series of novel koumine-like compounds were designed, semi-synthesized and systematically evaluated for antitumor activities.Methods: All compounds were evaluated for antiproliferative activity against four human cancer cell lines, including HT-29, HCT-116, HCT-15 and Caco-2.Results: Most compounds exhibited much higher antiproliferation activities (IC50<10μM) than koumine. Two selected compounds A4 and C5 showed comparable antitumor effects to 5-FU in vivo, as well as better safety profiles. Further studies suggested that A4 and C5 could arrest HT-29 cell cycle in G2 phase and raise reactive oxygen specieslevel, thus inducing cell apoptosis related to Erk MAPK and NF-κB signaling pathways inhibition.Conclusion: These results will greatly promote the druggability study of these koumine-like compounds.

Discovery of potent CSK inhibitors through integrated virtual screening and molecular dynamic simulation.

Oncogenic overexpression or activation of C-terminal Src kinase (CSK) has been shown to play an important role in triple-negative breast cancer (TNBC) progression, including tumor initiation, growth, metastasis, drug resistance. This revelation has pivoted the focus toward CSK as a potential target for novel treatments. However, until now, there are few inhibitors designed to target the CSK protein. Responding to this, our research has implemented a comprehensive virtual screening protocol. By integrating energy-based screening methods with AI-driven scoring functions, such as Attentive FP, and employing rigorous rescoring methods like Glide docking and molecular mechanics generalized Born surface area (MM/GBSA), we have systematically sought out inhibitors of CSK. This approach led to the discovery of a compound with a potent CSK inhibitory activity, reflected by an IC50 value of 1.6 nM under a homogeneous time-resolved fluorescence (HTRF) bioassay. Subsequently, molecule 2 exhibits strong growth inhibition of MD anderson - metastatic breast (MDA-MB) -231, Hs578T, and SUM159 cells, showing a level of growth inhibition comparable to that observed with dasatinib. Treatment with molecule 2 also induced significant G1 phase accumulation and cell apoptosis. Furthermore, we have explored the explicit binding interactions of the compound with CSK using molecular dynamics simulations, providing valuable insights into its mechanism of action.

Adenovirus-mediated expression of MOAP-1, Bax and RASSF1A antagonizes chemo-drug resistance of human breast cancer cells expressing cancer stem cell markers

Cancer is one of the major leading causes of mortality globally and chemo-drug-resistant cancers pose significant challenges to cancer treatment by reducing patient survival rates and increasing treatment costs. Although the mechanisms of chemoresistance vary among different types of cancer, cancer cells are known to share several hallmarks, such as their resistance to apoptosis as well as the ability of cancer stem cells to produce metastatic daughter cells that are resistant to chemotherapy. To address the issue of chemo-drug resistance in cancer cells, a tetracistronic expression construct, Ad-MBR-GFP, encoding adenovirus-mediated expression of MOAP-1, Bax, RASSSF1A, and GFP, was generated to investigate its potential activity in reducing or inhibiting the chemo-drug resistant activity of the human breast cancer cells, MCF-7-CR and MDA-MB-231. When infected by Ad-MBR-GFP, the cancer cells exhibited round cell morphology and nuclei condensation with positive staining for annexin-V. Furthermore, our results showed that both MCF-7-CR and MDA-MB-231 cells stained positively for CD 44 and negatively for CD 24 (CD44+/CD24-) with high levels of endogenous ALDH activity whereas SNU-1581 breast cancer cells were identified as CD 44-/CD 24- cells with relatively low levels of endogenous ALDH activity and high sensitivity toward chemo-drugs, suggesting that both CD 44 and ALDH activity contribute to chemo-drug resistance. Moreover, both MCF-7-CR and MDA-MB-231 cells showed strong chemo-drug sensitivity to cisplatin when the cells were infected by Ad-MBR-GFP, leading to 9-fold and 2-fold reduction in the IC 50 values when compared to cisplatin treatment alone, respectively. The data were further supported by 3D (soft agar) and spheroid cell models of MCF-7-CR and MDA-MB-231 cells which showed a 2-fold reduction of a number of cell colonies and spheroid size when treated with both Ad-MBR-GFP and cisplatin, and compared to control. Other than chemo-sensitivity, Ad-MBR-GFP-infected cancer cells retarded cell migration. Flow cytometry analysis showed that the mechanism of action of Ad-MBR-GFP involved cell cycle arrest at the G1 phase and inhibition of cellular DNA synthesis. Taken together, our investigation showed that Ad-MBR-GFP mediated chemo-drug sensitization in the infected cancer cells involved the activation of apoptosis signaling, cell cycle arrest, and inhibition of DNA synthesis, suggesting that Ad-MBR-GFP is potentially efficacious for the treatment of chemo-drug resistant cancers.

Time of exercise differentially impacts bone growth in mice.

Although physical training has been shown to improve bone mass, the time of day to exercise for optimal bone growth remains uncertain. Here we show that engaging in physical activity during the early active phase, as opposed to the subsequent active or rest phase, results in a more substantial increase in bone length of male and female mice. Transcriptomic and metabolomic methodologies identify that exercise during the early active phase significantly upregulates genes associated with bone development and metabolism. Notably, oxidative phosphorylation-related genes show a rhythmic expression in the chondrification centre, with a peak at the early active phase, when more rhythmic genes in bone metabolism are expressed and bone growth is synergistically promoted by affecting oxidative phosphorylation, which is confirmed by subsequent pharmacological investigations. Finally, we construct a signalling network to predict the impact of exercise on bone growth. Collectively, our research sheds light on the intricacies of human exercise physiology, offering valuable implications for interventions.

CDK2 and CDK4: Cell Cycle Functions Evolve Distinct, Catalysis-Competent Conformations, Offering Drug Targets.

Cyclin-dependent kinases (CDKs), particularly CDK4 and CDK2, are crucial for cell cycle progression from the Gap 1 (G1) to the Synthesis (S) phase by phosphorylating targets such as the Retinoblastoma Protein (Rb). CDK4, paired with cyclin-D, operates in the long G1 phase, while CDK2 with cyclin-E, manages the brief G1-to-S transition, enabling DNA replication. Aberrant CDK signaling leads to uncontrolled cell proliferation, which is a hallmark of cancer. Exactly how they accomplish their catalytic phosphorylation actions with distinct efficiencies poses the fundamental, albeit overlooked question. Here we combined available experimental data and modeling of the active complexes to establish their conformational functional landscapes to explain how the two cyclin/CDK complexes differentially populate their catalytically competent states for cell cycle progression. Our premise is that CDK catalytic efficiencies could be more important for cell cycle progression than the cyclin-CDK biochemical binding specificity and that efficiency is likely the prime determinant of cell cycle progression. We observe that CDK4 is more dynamic than CDK2 in the ATP binding site, the regulatory spine, and the interaction with its cyclin partner. The N-terminus of cyclin-D acts as an allosteric regulator of the activation loop and the ATP-binding site in CDK4. Integrated with a suite of experimental data, we suggest that the CDK4 complex is less capable of remaining in the active catalytically competent conformation, and may have a lower catalytic efficiency than CDK2, befitting their cell cycle time scales, and point to critical residues and motifs that drive their differences. Our mechanistic landscape may apply broadly to kinases, and we propose two drug design strategies: (i) allosteric Inhibition by conformational stabilization for targeting allosteric CDK4 regulation by cyclin-D, and (ii) dynamic entropy-optimized targeting which leverages the dynamic, entropic aspects of CDK4 to optimize drug binding efficacy.

Genome folding principles uncovered in condensin-depleted mitotic chromosomes.

During mitosis, condensin activity is thought to interfere with interphase chromatin structures. To investigate genome folding principles in the absence of chromatin loop extrusion, we codepleted condensin I and condensin II, which triggered mitotic chromosome compartmentalization in ways similar to that in interphase. However, two distinct euchromatic compartments, indistinguishable in interphase, emerged upon condensin loss with different interaction preferences and dependencies on H3K27ac. Constitutive heterochromatin gradually self-aggregated and cocompartmentalized with facultative heterochromatin, contrasting with their separation during interphase. Notably, some cis-regulatory element contacts became apparent even in the absence of CTCF/cohesin-mediated structures. Heterochromatin protein 1 (HP1) proteins, which are thought to partition constitutive heterochromatin, were absent from mitotic chromosomes, suggesting, surprisingly, that constitutive heterochromatin can self-aggregate without HP1. Indeed, in cells traversing from M to G1 phase in the combined absence of HP1α, HP1β and HP1γ, constitutive heterochromatin compartments are normally re-established. In sum, condensin-deficient mitotic chromosomes illuminate forces of genome compartmentalization not identified in interphase cells.

Synthesis, Antiproliferative Activity and Molecular Docking Analysis of Both Enantiomerically Pure Decursin Derivatives as Anticancer Agents.

Using (S)-decursinol isolated from root of Angelica gigas Nakai (AGN), we semi-synthesized and evaluated a series of both enantiomerically pure decursin derivatives for their antiproliferative activities against A549 human lung cancer cells. All synthesized compounds showed a broad spectrum of inhibitory activities against the growth of A549 cells. Especially, compound (S)-2d with (E)-(furan-3-yl)acryloyl group showed the most potent activity (IC50: 14.03 µM) against A549 cancer cells as compared with the reference compound, decursin (IC50: 43.55 µM) and its enantiomer, (R)-2d (IC50: 151.59 µM). Western blotting assays indicated that (S)-2d more strongly inhibited Janus kinase 1 (JAK1) and signal transducer and activator of transcription activation 3 (STAT3) phosphorylation than decursin in a dose-dependent manner, while having no effect on CXCR7 overexpression and total STAT3 level. In addition, (S)-2d induced cell cycle arrest at G1 phase and subsequent apoptotic cell death in A549 cancer cells. Our combined analysis of molecular docking studies and biological data suggests that the inhibition of JAK1 with (S)-2d resulted in loss of STAT3 phosphorylation and inhibition of cell growth in A549 cancer cells. These overall results strongly suggest that (S)-2d (MRC-D-004) as a novel JAK1 inhibitor may have therapeutic potential in the treatment of A549 human lung cancers by targeting the JAK1/STAT3 signaling pathway.

Circadian rhythm in pro-arrhythmic activity of pulmonary vein cardiomyocytes

Abstract Introduction Cardiomyocytes in the pulmonary vein (PV) sleeves are a major source of ectopic activity driving atrial fibrillation (AF) and episodes of AF in patients are more prevalent at night. While it is known that circadian clocks within the heart regionally control 24-hour variation in pacemaking and ventricular repolarisation, the mechanisms underlying the nighttime preponderance in AF are unknown. Purpose This study addresses the hypothesis that circadian rhythms exist in the pro-arrhythmic activity of PV cardiomyocytes. Methods Male Wistar rats maintained in a 24-hour cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT=0; lights-off, ZT12) were subject to terminal general anaesthesia (140 mg/kg Na pentobarbital i.p.) at ZT=0, 6, 12 or 18 hr and the hearts removed. RNA was extracted from left atrial (LA) appendage (LAA) and proximal PV at the LA/PV junction (3 rats per ZT) and RNA sequencing conducted. Reads were mapped to the rat genome, counts normalised and models in which ZT was or was not included as a factor compared (Likelihood Ratio Test, adjusted-P&amp;lt;0.01). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=308) and the LAA (n=264) (N=74 rats). The effects of noradrenaline (NA, 1 µM) and acetylcholine (ACh, 1 µM) were examined. Data were plotted against the ZT of the time of recording and fitted to a sine wave to establish circadian rhythmicity (P&amp;lt;0.05, extra-sum-of-squares F-test). The effect of ≥24 hr constant dark in the period immediately before experiment was examined. Data are reported as mean ± standard error. Results The expression of 1368 genes varied significantly with ZT, including circadian clock components (e.g. Bmal1, Per1). PV cells were larger than LAA cells (73±1.6 pF vs 53±1.3 pF, P&amp;lt;0.0001) and had more depolarised resting membrane potential (-69±0.2 mV vs -72±0.1 mV, P&amp;lt;0.0001). Both cell types showed circadian variation in action potential duration at 90% repolarisation (APD90) and frequency of pro-arrhythmic activity. Pro-arrhythmic activity was greatest in PV cells and the frequency was greater during the rest phase (ZT0-12) in both cell types. In contrast, the circadian rhythm in APD90 differed between cell type, with the longest APD­­90 recorded during the active phase in PV cells (ZT12-24) but during the rest phase in LAA cells. Pro-arrhythmic activity was increased by NA and decreased by ACh in both cell types with maximal effect during the rest phase. Conclusion Circadian variation in APD90 and proarrhythmic-activity has been demonstrated in isolated proximal PV and LAA cardiomyocytes, with differences in rhythm between the two cell types. RNA sequencing suggests the presence of peripheral clocks in LAA and PV cardiomyocytes. Understanding these mechanisms is important in development of future therapeutic options for AF.

Exercise-induced regional heterogeneity of myocardial left ventricular contraction in hereditary long QT syndrome

Abstract Background Hereditary long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization and an increased risk for ventricular arrhythmias (VA) and sudden cardiac death. Dispersion of ventricular repolarization in LQTS may be more pronounced during exercise and not only impacts the heart’s electrical functions but also the pattern of mechanical contraction. Little is known about the intraventricular heterogeneity of wall motion abnormalities in LQTS patients. Methods In this cross-sectional study we quantify regional, transmural, and longitudinal alterations in left ventricular (LV) contraction at rest, during exercise, and recovery in genetically confirmed LQTS patients and healthy controls to evaluate their value in differentiating high risk patients as defined by a history of VA. ECG and echocardiographic assessments were conducted during rest, peak ergometer activity (105 to 125 bpm), and four minutes into recovery phases. Longitudinal strain (LStr) parameters were measured for each LV segment separately. We differentiated LStr variations as heterogeneity of LStr amplitude (hetLStr; standard deviation [SD] or mean range) and dispersion of time-to-peak LStr (disLStr; SD or mean range). Here, we present the analyses of disLStr. Results At present, 15 patients with genetically confirmed LQTS (LQT1: 8, LQTS 2: 7; mean QTc 475 ± 46 ms) have been included of which 9 had a history of VA. In addition, we collected data from 8 age- and gender-matched control patients (mean QTc 401 ± 18 ms). LV ejection fraction and LStr were not significantly different between the LQTS and control groups. However, LQTS patients exhibited a significantly lower mean LStr in both the endocardial and epicardial layers across all phases compared to control. We observed a significantly more pronounced disLStr across all segments in the LQTS group during the rest and recovery phases in the endocardial layer and during all phases in the epicardial layer. Regional differences (apical vs. basal) were found in all LQTS patients only endocardially at rest. Globally, disLStr showed transmural differences (epicardial vs. endocardial) in both asymptomatic and symptomatic LQTS patients during peak exercise and recovery. Upon segmental analysis, it was observed that symptomatic patients exhibited these transmural differences apically already at rest (p=.004), as well as in the basal region during peak exercise (p=.01), and in the apical region during recovery (p=.02). Conclusion LQTS patients show regional (base to apex) and transmural (endocardial to epicardial) heterogeneities in myocardial contraction at rest, during exercise, and recovery as compared to healthy patients. These regional variations in myocardial contraction patterns may be more pronounced in high risk LQTS patients. Ongoing analyses of hetLStr are in progress to corroborate our findings.

Identification of novel GSPT1 degraders by virtual screening and bioassay

GSPT1 plays crucial physiological functions, such as terminating protein translation, overexpressed in various tumors. It is a promising anti-tumor target, but is also considered as an “undruggable” protein. Recent studies have found that a class of small molecules can degrade GSPT1 through the “molecular glue” mechanism with strong antitumor activity, which is expected to become a new therapy for hematological malignancies. Currently available GSPT1 degraders are mostly derived from the scaffold of immunomodulatory imide drug (IMiD), thus more active compounds with novel structure remain to be found. In this work, using computer-assisted multi-round virtual screening and bioassay, we identified a non-IMiD acylhydrazone compound, AN5782, which can reduce the protein level of GPST1 and obviously inhibit the proliferation of tumor cells. Some analogs were obtained by a substructure search of AN5782. The structure-activity relationship analysis revealed possible interactions between these compounds and CRBN-GSPT1. Further biological mechanistic studies showed that AN5777 decreased GSPT1 remarkably through the ubiquitin-proteasome system, and its effective cytotoxicity was CRBN- and GSPT1-dependent. Furthermore, AN5777 displayed good antiproliferative activities against U937 and OCI-AML-2 cells, and dose-dependently induced G1 phase arrest and apoptosis. The structure found in this work could be good start for antitumor drug development.

#2936 Chronic kidney disease in systemic sclerosis

Abstract Background and Aims Systemic sclerosis is a heterogeneous autoimmune disease with multiorgan involvement. To assess the clinical and epidemiological manifestations of patients with SSc who present CKD, and its implication in the prognosis and its importance in early detection. Method Selected 102 Caucasian patients (12 men and 90 women) diagnosed with SSc who were seen consecutively in the Scleroderma Unit at Vall d'Hebron Hospital since 1980. The following demographic variables were collected: sex, age, common cardiovascular risk factors (diabetes mellitus, hypertension, dyslipidemia), age at clinical onset, age at diagnosis, skin subtype of SSc according to the LeRoy and Medsger classification, visceral conditions, osteoarticular conditions, capillaroscopic pattern, biochemical parameters of renal function and immunological alterations, renal morphological alterations by ultrasound, and treatments. Results Overall, 102 SSc patients were included, of whom 24 (23.5%) had renal failure. The mean plasma creatinine value was 0.92 mg/dl and the mean GFR by CKD-EPI was 74.80 ml/min. The 76.5% had GFRe ≥60 ml/min (SScCKD-), with mean serum creatinine 0.85 ± 0.17 mg/dl and mean GFRe 82.13 ml/min (SD 20.53 ml/min); 23.5% had GFRe &amp;lt;60 ml/min (SScCKD+), in them mean serum creatinine was 1.09 ± 0.15 mg/dl and mean GFRe was 51.29 ml/min (SD 6.68 ml/min). By stage: 18 patients had G1 stage, 60 patients had G2 stage, 18 patients had GFR 3a and 6 patients GFR G3b. No proteinuria or hematuria was detected in any of the patients. In univariate analysis, limited cutaneous subtype was significantly more frequent in the SScCKD+ group (20, 83.3% vs. 44, 56.4%, p = 0.017) and the diffuse cutaneous subtype was more frequent in the SScCKD- group (0, 0% vs. 23, 29.4%, p &amp;lt; 0.001). In the study of associated antibodies, ACAs (20, 83.3% vs. 36, 46.1%, p = 0.001) were statistically significantly more frequent in the SScCKD+ group. No ATA were detected in SScCKD+ patients while in the SScCKD- group 25.6% had ATA (p = 0.003). Pulmonary involvement in the form of PAH was significantly more frequent in the SScCKD+ group (13, 54.1% vs. 19, 24.3%, p&amp;lt; 0.05). In pulmonary involvement in the form of ILD no differences were found (9, 37.5% vs. 37, 47.4% in SScCKD+ and SScCKD- patients, respectively). Regarding PFR results, there were no differences in the percentage of patients with FVC below 70% or 50% of the expected value (6, 25% vs. 15, 19.2% and 1, 4.1% vs. 4, 5.1%, respectively). Significant differences were found in mortality in the two groups (12, 50% vs. 16, 20.5%, in the SScCKD+ and SScCKD- group, respectively, p &amp;lt; 0.05), but not in the causes of death. Conclusion The presence of CKD in SSc conferred a clinical profile and prognosis.

Palbociclib-derived multifunctional molecules for lysosomal targeting and diagnostic-therapeutic integration.

Aim: Lysosomal pH changes are associated with drug resistance, cell growth and invasion of tumors, but effective and specific real-time monitoring of lysosomal pH compounds for cancer therapy is lacking. Materials & methods: Here, based on the covalent linkage of the anticancer drug palbociclib and fluorescent dyefluorescein isothiocyanate (FITC), we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Results & discussion: Pal-FITC fluoresces is 20-fold stronger than that of FITC and shows a linear response in the pH range of 4.0-8.2 (R2=0.9901). Pal-FITC blocks cells in G1 phase via Cyclin D-CDK4/6-Rb. Conclusion: Our study provides new strategies for tumor-targeted imaging and personalized therapy.

SMYD2-Methylated PPARγ Facilitates Hypoxia-Induced Pulmonary Hypertension by Activating Mitophagy.

Hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) and consequent pulmonary vascular remodeling are the crucial pathological features of pulmonary hypertension (PH). Protein methylation has been shown to be critically involved in PASMC proliferation and PH, but the underlying mechanism remains largely unknown. PH animal models were generated by treating mice/rats with chronic hypoxia for 4 weeks. SMYD2-vTg mice (vascular smooth muscle cell-specific suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 (deformed epidural auto-regulatory factor-1) domain-containing protein 2 transgenic) or wild-type rats and mice treated with LLY-507 (3-cyano-5-{2-[4-[2-(3-methylindol-1-yl)ethyl]piperazin-1-yl]-phenyl}-N-[(3-pyrrolidin-1-yl)propyl]benzamide) were used to investigate the function of SMYD2 (suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2) on PH development in vivo. Primary cultured rat PASMCs with SMYD2 knockdown or overexpression were used to explore the effects of SMYD2 on proliferation and to decipher the underlying mechanism. We demonstrated that the expression of the lysine methyltransferase SMYD2 was upregulated in the smooth muscle cells of pulmonary arteries from patients with PH and hypoxia-exposed rats/mice and in the cytoplasm of hypoxia-induced rat PASMCs. More importantly, targeted inhibition of SMYD2 by LLY-507 significantly attenuated hypoxia-induced pulmonary vascular remodeling and PH development in both male and female rats in vivo and reduced rat PASMC hyperproliferation in vitro. In contrast, SMYD2-vTg mice exhibited more severe PH phenotypes and related pathological changes than nontransgenic mice after 4 weeks of chronic hypoxia treatment. Furthermore, SMYD2 overexpression promoted, while SMYD2 knockdown suppressed, the proliferation of rat PASMCs by affecting the cell cycle checkpoint between S and G2 phases. Mechanistically, we revealed that SMYD2 directly interacted with and monomethylated PPARγ (peroxisome proliferator-activated receptor gamma) to inhibit the nuclear translocation and transcriptional activity of PPARγ, which further promoted mitophagy to facilitate PASMC proliferation and PH development. Furthermore, rosiglitazone, a PPARγ agonist, largely abolished the detrimental effects of SMYD2 overexpression on PASMC proliferation and PH. Our results demonstrated that SMYD2 monomethylates nonhistone PPARγ and inhibits its nuclear translocation and activation to accelerate PASMC proliferation and PH by triggering mitophagy, indicating that targeting SMYD2 or activating PPARγ are potential strategies for the prevention of PH.

Cell division cyclin 25C knockdown inhibits hepatocellular carcinoma development by inducing endoplasmic reticulum stress.

Cell division cyclin 25C (CDC25C) is a protein that plays a critical role in the cell cycle, specifically in the transition from the G2 phase to the M phase. Recent research has shown that CDC25C could be a potential therapeutic target for cancers, particularly for hepatocellular carcinoma (HCC). However, the specific regulatory mechanisms underlying the role of CDC25C in HCC tumorigenesis and development remain incompletely understood. To explore the impact of CDC25C on cell proliferation and apoptosis, as well as its regulatory mechanisms in HCC development. Hepa1-6 and B16 cells were transduced with a lentiviral vector containing shRNA interference sequences (LV-CDC25C shRNA) to knock down CDC25C. Subsequently, a xenograft mouse model was established by subcutaneously injecting transduced Hepa1-6 cells into C57BL/6 mice to assess the effects of CDC25C knockdown on HCC development in vivo. Cell proliferation and migration were evaluated using a Cell Counting Kit-8 cell proliferation assays and wound healing assays, respectively. The expression of endoplasmic reticulum (ER) stress-related molecules (glucose-regulated protein 78, X-box binding protein-1, and C/EBP homologous protein) was measured in both cells and subcutaneous xenografts using quantitative real-time PCR (qRT-PCR) and western blotting. Additionally, apoptosis was investigated using flow cytometry, qRT-PCR, and western blotting. CDC25C was stably suppressed in Hepa1-6 and B16 cells through LV-CDC25C shRNA transduction. A xenograft model with CDC25C knockdown was successfully established and that downregulation of CDC25C expression significantly inhibited HCC growth in mice. CDC25C knockdown not only inhibited cell proliferation and migration but also significantly increased the ER stress response, ultimately promoting ER stress-induced apoptosis in HCC cells. The regulatory mechanism of CDC25C in HCC development may involve the activation of ER stress and the ER stress-induced apoptosis signaling pathway.