P21 Expression Research Articles

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by β-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by β-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

FAM13A regulates cellular senescence marker p21 and mitochondrial reactive oxygen species production in airway epithelial cells.

Inhalation of noxious gasses induces oxidative stress in airway epithelial cells (AECs), which may lead to cellular senescence and contribute to the development of Chronic Obstructive Pulmonary Disease (COPD). FAM13A, a well-known COPD susceptibility gene, is highly expressed in airway epithelium. We studied whether its expression is associated with aging and cellular senescence and affects airway epithelial responses to paraquat, a cellular senescence inducer. The association between age and FAM13A expression was investigated in two datasets of human lung tissue and bronchial brushings from current/ex-smokers with/without COPD. Protein levels of FAM13A and cellular senescence marker p21 were investigated using immunohistochemistry in lung tissue from COPD patients. In vitro, FAM13A and P21 expression was assessed using qPCR in air-liquid-interface (ALI)-differentiated AECs in absence/presence of paraquat. Additionally, FAM13A was overexpressed in human bronchial epithelial 16HBE cells and the effect on P21 expression (qPCR) and mitochondrial ROS production (MitoSOX staining) was assessed. Lower FAM13A expression was significantly associated with increasing age in lung tissue and bronchial epithelium. In airway epithelium of COPD patients, we found a negative correlation between FAM13A and p21 protein levels. In ALI-differentiated AECs, the paraquat-induced decrease in FAM13A expression was accompanied by increased P21 expression. In 16HBE cells, the overexpression of FAM13A significantly reduced paraquat-induced P21 expression and mitochondrial ROS production. Our data suggest that FAM13A expression decreases with aging, resulting in higher P21 expression and mitochondrial ROS production in the airway epithelium, thus facilitating cellular senescence and as such potentially contributing to accelerated lung aging in COPD.

CSE1L is a negative regulator of the RB-DREAM pathway in p53 wild-type NSCLC and can be targeted using an HDAC1/2 inhibitor

P53 represses transcription by activating p21 expression and promoting formation of RB1-E2F1 and RBL1/RBL2-DREAM transcription repressor complexes. The DREAM complex is composed of DP1, RB-family proteins RBL1 or RBL2 (p107/p130), E2F4/5, and MuvB. We recently reported RBL2-DREAM contributes to improved therapy responses in p53 wild-type NSCLC cells and improved outcomes in NSCLC patients whose tumors express wild-type p53. In the current study we identified CSE1L as a novel inhibitor of the RBL2-DREAM pathway and target to activate RBL2-DREAM in NSCLC cells. CSE1L is an oncoprotein that maintains repression of genes that can be reactivated by HDAC inhibitors. Mocetinostat is a HDAC inhibitor in clinical trials with selectivity against HDACs 1 and 2. Knockdown of CSE1L in NSCLC cells or treatment with mocetinostat increased p21, activated RB1 and RBL2, repressed DREAM target genes, and induced toxicity in a manner that required wild-type p53. Lastly, we found high levels of CSE1L and specific DREAM-target genes are candidate markers to identify p53 wild-type NSCLCs most responsive to mocetinostat. Thus, we identified CSE1L as a critical negative regulator of the RB-DREAM pathway in p53 wild-type NSCLC that can be indirectly targeted with HDAC1/2 inhibitors (mocetinostat) in current clinical trials. High expression of CSE1L and DREAM target genes could serve as a biomarker to identify p53 wild-type NSCLCs most responsive to this HDAC1/2 inhibitor.

Modulation of Viability, Proliferation, and Stemness by Rosmarinic Acid in Medulloblastoma Cells: Involvement of HDACs and EGFR.

Medulloblastoma (MB) is a heterogeneous group of malignant pediatric brain tumors, divided into molecular groups with distinct biological features and prognoses. Currently available therapy often results in poor long-term quality of life for patients, which will be afflicted by neurological, neuropsychiatric, and emotional sequelae. Identifying novel therapeutic agents capable of targeting the tumors without jeopardizing patients' quality of life is imperative. Rosmarinic acid (RA) is a plant-derived compound whose action against a series of diseases including cancer has been investigated, with no side effects reported so far. Previous studies have not examined whether RA has effects in MB. Here, we show RA is cytotoxic against human Daoy (IC50 = 168μM) and D283 (IC50 = 334μM) MB cells. Exposure to RA for 48h reduced histone deacetylase 1 (HDAC1) expression while increasing H3K9 hyperacetylation, reduced epidermal growth factor (EGFR) expression, and inhibited EGFR downstream targets extracellular-regulated kinase (ERK)1/2 and AKT in Daoy cells. These modifications were accompanied by increased expression of CDKN1A/p21, reduced expression of SOX2, and a decrease in proliferative rate. Treatment with RA also reduced cancer stem cell markers expression and neurosphere size. Taken together, our findings indicate that RA can reduce cell proliferation and stemness and induce cell cycle arrest in MB cells. Mechanisms mediating these effects may include targeting HDAC1, EGFR, and ERK signaling, and promoting p21 expression, possibly through an increase in H3K9ac and AKT deactivation. RA should be further investigated as a potential anticancer agent in experimental MB.

P21 promotes gemcitabine tolerance in A549 cells by inhibiting DNA damage and altering the cell cycle.

Gemcitabine is one of the most widely used chemotherapy drugs for advanced malignant tumors, including non-small cell lung cancer. However, the clinical efficacy of gemcitabine is limited due to drug resistance. The aim of the present study was to investigate the role of p21 in gemcitabine-resistant A549 (A549/G+) lung cancer cells. IC50 values were determined using a Cell Counting Kit-8 (CCK-8) assay. mRNA and protein expression levels of genes were measured by reverse transcription-quantitative PCR and western blotting, respectively. The cell cycle distribution and apoptosis rate were analyzed by flow cytometry. DNA damage in cells was evaluated by single-cell gel electrophoresis. The results of western blot analysis and the CCK-8 assay demonstrated that the expression of p21 was higher in A549/G+ cells than in gemcitabine-sensitive cells. Knockdown of p21 expression in gemcitabine-resistant cells sensitized these cells to gemcitabine (with the IC50 decreasing from 84.2 to 26.7 µM). Cell cycle analysis revealed different changes in the cell cycle distribution in A549/G+ cells treated with the same concentration of gemcitabine, and decreased expression of p21 was shown to promote G1 arrest. The apoptosis assay and comet assay results revealed that decreased p21 expression resulted in accumulation of unrepaired DNA double-strand breaks (DSBs) and induction of apoptosis by gemcitabine. The present study demonstrated that knockout of p21 mRNA expression in A549/G+ cells promotes apoptosis and DNA DSB accumulation, accompanied by G1 arrest. These results indicated that p21 is involved in regulating the response of A549 cells to gemcitabine.

Erythritol Can Inhibit the Expression of Senescence Molecules in Mouse Gingival Tissues and Human Gingival Fibroblasts.

Oral aging causes conditions including periodontal disease. We investigated how the sugar alcohol erythritol, which has anti-caries effects, impacts aging periodontal tissues and gingival fibroblasts in mice and humans in vivo and in vitro. Mice were classified into three groups: control groups of six-week-old (YC) and eighteen-month-old mice (AC) and a group receiving 5% w/w erythritol water for 6 months (AE). After rearing, RNA was extracted from the gingiva, and the levels of aging-related molecules were measured using PCR. Immunostaining was performed for the aging markers p21, γH2AX, and NF-κB p65. p16, p21, γH2AX, IL-1β, and TNFα mRNA expression levels were higher in the gingiva of the AC group than in the YC group, while this enhanced expression was significantly suppressed in AE gingiva. NF-κB p65 expression was high in the AC group but was strongly suppressed in the AE group. We induced senescence in cultured human gingival fibroblasts using H2O2 and lipopolysaccharide before erythritol treatment, which reduced elevated senescence-related marker (p16, p21, SA-β-gal, IL-1β, and TNFα) expression levels. Knockdown of PFK or PGAM promoted p16 and p21 mRNA expression, but erythritol subsequently rescued pyruvate production. Overall, intraoral erythritol administration may prevent age-related oral mucosal diseases.

P21 Prevents the Exhaustion of CD4+ T Cells Within the Antitumor Immune Response Against Colorectal Cancer

Background and aimsT cells are crucial for the antitumor response against colorectal cancer (CRC). T cell reactivity to CRC is nevertheless limited by T cell exhaustion. However, molecular mechanisms regulating T cell exhaustion are only poorly understood. MethodsWe investigated the functional role of cyclin-dependent kinase 1a (Cdkn1a or p21) in CD4+ T cells using murine CRC models. Furthermore, we evaluated the expression of p21 in patients with stage I–IV CRC. In vitro co-culture models were used to understand the effector function of p21-deficient CD4+ T cells. ResultsWe observed that the activation of cell cycle regulator p21 is crucial for CD4+ T cell cytotoxic function and that p21-deficiency in Th1 cells leads to increased tumor growth in murine CRC. Similarly, low p21 expression in CD4+ T cells infiltrated into tumors of CRC patients is associated with reduced cancer-related survival. In mouse models of CRC, p21-deficient Th1 cells show signs of exhaustion, where an accumulation of effector/effector memory T cells and CD27/CD28 loss are predominant. Immune reconstitution of tumor-bearing Rag1-/- mice using ex vivo treated p21-deficient T cells with Palbociclib, an inhibitor of CDK4/6, restored cytotoxic function and prevented exhaustion of p21-deficient CD4+ T cells as a possible concept for future immunotherapy of human disease. ConclusionOur data reveal the importance of p21 in controlling cell cycle and preventing exhaustion of Th1 cells. Furthermore, we unveil the therapeutic potential of CDK inhibitors such as Palbociclib to reduce T cell exhaustion for future treatment of patients with colorectal cancer.

New role for the anandamide metabolite prostaglandin F2α ethanolamide: Rolling preadipocyte proliferation

White adipose tissue regulation is key to metabolic health, yet still perplexing. The chief endocannabinoid anandamide metabolite, prostaglandin F2α ethanolamide (PGF2αEA), inhibits adipogenesis, that is, the formation of mature adipocytes. We observed that adipocyte progenitor cells—preadipocytes—following treatment with PGF2αEA yielded larger pellet sizes. Thus, we hypothesized that PGF2αEA might augment preadipocyte proliferation. Cell viability MTT and crystal violet assays, cell counting, and 5-bromo-2′-deoxyuridine incorporation in cell proliferation ELISA analyses confirmed our prediction. Additionally, we discovered that PGF2αEA promotes cell cycle progression through suppression of the expression of cell cycle inhibitors, p21 and p27, as shown by flow cytometry and qPCR. Enticingly, concentrations of this compound that showed no visible effect on cell proliferation or basal transcriptional activity of peroxisome proliferator-activated receptor gamma could, in contrast, reverse the anti-proliferative and peroxisome proliferator-activated receptor gamma-transcription activating effects of rosiglitazone (Rosi). MTT and luciferase reporter examinations supported this finding. The PGF2αEA pharmaceutical analog, bimatoprost, was also investigated and showed very similar effects. Importantly, we suggest the implication of the mitogen-activated protein kinase pathway in these effects, as they were blocked by the selective mitogen-activated protein kinase kinase inhibitor, PD98059. We propose that PGF2αEA is a pivotal regulator of white adipose tissue plasticity, acting as a regulator of the preadipocyte pool in adipose tissue.

HPF1 regulates tendon stem/progenitor cell senescence and tendon repair via PARP1-mediated poly-ADP ribosylation of HuR.

Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated β-galactosidase (SA-β-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.

Aberrant DNA methylation of CDKN2B gene in the Type 1 diabetes mellitus and analysis of epigenetic markers

IntroductionAberrant DNA methylation is one of the important molecular markers in acute lymphoblastic leukemia (ALL). ObjectivesTo determine Aberrant DNA methylation profiles of the CDKN2A and CDKN2B genes in ALL samples from T1DM cases. ResultsAberrant hypermethylation of CDKN2B gene and cytogenetic abnormalities were analyzed in >60% of subjected cases. Total cholesterol, triglycerides and low-density lipoprotein cholesterol were not significantly varied between experimental and control (p >0.05). Expression analysis of human peripheral blood mononuclear cells revealed decreased mRNA levels of CDKN2B. The decreased level of p21 and CDK4 expression in the T1DM cases than control. The number of monocytes, lymphocytes and neutrophils significantly varied in T1DM samples and control (p < 0.05). ConclusionsAberrant methylation is one of the important mechanisms involved in the expression of CDKN2B genes and is an important prognostic marker for the early detection of ALL risks.

Rumex vesicarius L. boosts the effectiveness of sorafenib in triple-negative breast cancer by downregulating BCl2, mTOR, and JNK, and upregulating p21 expression

Background/AimTriple-negative breast cancer (TNBC) is characterized by poor prognosis, rapid progression, serious clinical behavior, an elevated risk of metastasis, and resistance to standard treatments. Traditional medicine practitioners value Rumex vesicarius L. (RMV) for a variety of reasons, including the plant's antioxidant capabilities. Our study's goals were to ascertain the efficacy of RMV alone and in combination with sorafenib (SOR) against the aggressive TNBC cell line (MDA-MB-231) and use in vitro and in silico analysis to deduce the fundamental mechanism of action. MethodsIn the current study, molecular operating environment (MOE, 2019.0102) software was used for performing molecular docking. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to determine the cytotoxicity of RMV, SOR or RMV/SOR combination against the TNBC cell line MDA-MB-231 cells. The effects of RMV, SOR, and RMV and SOR combining on mRNAs expressions of the target genes including mTOR, p21, JNK, and BCl2 were evaluated. In TNBC cells, the relative expressions of mRNAs of the genes were examined by using real-time quantitative polymerase chain reaction (RT-qPCR). ResultsIn our experiments, we discovered that both RMV extracts alone and in combination with SOR considerably reduced cancer cell proliferation (IC50 = 0.83 and 0.19 μM, respectively). Additionally, the expression of the tumor suppressor gene p21 was elevated whereas the expression of the invasion and anti-apoptosis genes BCl2, mTOR, and JNK were significantly decreased after treatment with RMV and SOR. Based on in silico analysis, it was found that RMV extract contains bioactive chemicals with a high affinity for inhibiting JNK and VEGFR-2. ConclusionIn conclusion, in vitro and in silico investigations show that the RMV extract improves the anticancer efficiency of SOR through molecular processes involving the downregulation of mTOR, BCl2, and JNK1 and overexpression of p21 tumor suppressor gene. Finally, we suggest conducting additional in vivo investigations on RMV and its bioactive components to verify their potential in cancer therapy.

EXOSC10 is a novel hepatocellular carcinoma prognostic biomarker: a comprehensive bioinformatics analysis and experiment verification.

Hepatocellular carcinoma (HCC) is a common malignant tumor. There are few studies on EXOSC10 (exosome component 10) in HCC; however, the importance of EXOSC10 for HCC remains unclear. In the study, the prognosis value of EXOSC10 and the immune correlation were explored by bioinformatics. The expression of EXOSC10 was verified by tissue samples from clinical patients and in vitro experiment (liver cancer cell lines HepG2, MHCC97H and Huh-7; normal human liver cell line LO2). Immunohistochemistry (IHC) was used to detect EXOSC10 protein expression in clinical tissue from HCC. Huh-7 cells with siEXOSC10 were constructed using lipofectamine 3000. Cell counting kit 8 (CCK-8) and colony formation were used to test cell proliferation. The wound healing and transwell were used to analyze the cell migration capacity. Mitochondrial membrane potential, Hoechst 33342 dye, and flow cytometer were used to detect the change in cell apoptosis, respectively. Differential expression genes (DEGs) analysis and gene set enrichment analysis (GSEA) were used to investigate the potential mechanism of EXOSC10 and were verified by western blotting. EXOSC10 was highly expressed in tissues from patients with HCC and was an independent prognostic factor for overall survival (OS) in HCC. Increased expression of EXOSC10 was significantly related to histological grade, T stage, and pathological stage. Multivariate analysis indicated that the high expression level of EXOSC10 was correlated with poor overall survival (OS) in HCC. GO and GSEA analysis showed enrichment of the cell cycle and p53-related signaling pathway. Immune analysis showed that EXOSC10 expression was a significant positive correlation with immune infiltration in HCC. In vitro experiments, cell proliferation and migration were inhibited by the elimination of EXOSC10. Furthermore, the elimination of EXOSC10 induced cell apoptosis, suppressed PARP, N-cadherin and Bcl-2 protein expression levels, while increasing Bax, p21, p53, p-p53, and E-cadherin protein expression levels. EXOSC10 had a predictive value for the prognosis of HCC and may regulate the progression of HCC through the p53-related signaling pathway.

Abstract P178: Role Of The C-C Motif Chemokine Receptor 5 (CCR5) Polymorphism In A Mouse Model Of Kawasaki Disease-Induced Vasculitis

Introduction: Kawasaki disease (KD) is the most common form of vasculitis in young children. However, the etiology and the vulnerability of vasculitis are still enigmatic. In children, polymorphisms in CCR5 influence KD susceptibility and cardiovascular outcome, but the mechanisms are unclear. Therefore, the use of murine models is an attractive strategy to evaluate the interface between immune system and vasculitis in KD. We aimed to explore the mechanisms by which polymorphisms in CCR5 aggravates the vascular injury in KD by using the Candida albicans water soluble (CAWS) fraction model. Methods: Male (6-10-weeks-old) CCR5 deficient mice (CCR5 -/- ) and their counterpart (CCR5 +/+ ) were used. Results: After 28 days of CAWS treatment, CCL5 and CCR5 levels were increased in plasma [(pg/mL) CCR5 +/+ : 5.01 ± 0.7 n=5 vs . CCR5 +/+ CAWS: 18.71 ± 1.4* n=4, *p&lt;0.05], and heart (14-fold changes) of CCR5 +/+ , which was associated with a clear vasculitis in coronary and AA, as well as cardiac senescence (p21 expression), cell death (caspase-3 levels), and higher contractility to norepinephrine (NA) in AA [(mN) CCR5 +/+ : 3.60 ± 0.3 n=4 vs . CCR5 +/+ CAWS: 5.72 ± 0.2* n=4, *p&lt;0.05]. Remarkably, CCR5 -/- displayed a worse vasculitis level, further accumulation of cardiac p21 and caspase-3, and an exacerbated vascular response to NA (CCR5 -/- -CAWS: 6.70 ± 0.4 # n=5, # p&lt;0.05 vs . CCR5 +/+ CAWS). To understand the mechanisms, we performed a plasma proteome profile cytokine assay, which revealed that KD elevates the levels of various inflammatory mediators (IL-1β, IL-6, IL-17, and TNFα) and chemokines (CXCL2, 10, 11, 19, 12, and 13 and CCL2, 3, and 4), which were exacerbated in CCR5 -/- . Yet, CAWS induced an increase in cardiac neutrophil infiltration in CCR5 +/+ [(% in relation to 10 ^ 6 leukocytes) CCR5 +/+ : 2.5 ± 1.7 n=4 vs . CCR5 +/+ CAWS: 16.1 ± 3.2* n=4, *p&lt;0.05], which was worsened in CCR5 -/- (CCR5 -/- -CAWS: 30.2 ± 7.4 # n=4, # p&lt;0.05 vs . CCR5 +/+ CAWS). Conclusion: Deficiency in CCR5 aggravates the vasculitis levels and cardiovascular outcomes likely regulating neutrophil infiltration and cytokines production. Therefore, children with polymorphism in CCR5 might be more susceptible in developing a severe vasculitis during KD.

Rh(III) and Ru(II) complexes with phosphanyl-alkylamines: inhibition of DNA synthesis induced by anticancer Rh complex.

Aim: This investigation was designed to synthesize half-sandwich Rh(III) and Ru(II) complexes and study their antiproliferative activity in human cancer cell lines. Materials & methods: Nine compounds were prepared and tested by various assays for their anticancer activity and mechanism of action. Results: Hit Rh(III) complex 6 showed low-micromolar potency in cisplatin-sensitive (A2780) and -resistant (A2780cis) ovarian carcinoma cell lines, promising selectivity toward these cancer cells over normal lung fibroblasts and anunprecedented mechanism of action in the treated cells. DNA synthesis was decreased andCDKN1A expression was upregulated, but p21 expression was not induced. Conclusion: Rh complex 6 showed high antiproliferative activity, which is induced through a p21-independent mechanism of action.

Design, synthesis and biological evaluation of pyrimidine base hydroxamic acid derivatives as dual JMJD3 and HDAC inhibitors

The Jumonji domain-containing protein demethylase 3 (JMJD3) and histone deacetylase (HADC) are related to various cancers and regard as antitumor targets for drug discovery. In this study, based on rational drug design strategy, we designed and synthesized a series of pyrimidine derivatives with hydroxamic acid as novel dual JMJD3 and HDAC inhibitors for synergistic cancer treatment. Compound A5b exhibited inhibitory potency against JMJD3 and HDAC1/6 simultaneously and favorable cytotoxicity against human cancer cells such as A549 and U937. Furthermore, mechanistic studies showed that A5b treatment in A549 cells increased the hypermethylation of histone H3K27 and hyperacetylation of H3K9, suppressed clonogenicity, migration and invasion of cancer cells. Besides, A5b induced apoptosis via the cleavage of caspase-7 and PARP, and G1 cell cycle arrest via upregulated p21 expression. All these results suggested that A5b was the first dual inhibitor against JMJD3 and HDAC and can be a potential compound for cancer therapy.

Regulation of HDAC11 gene expression in early myogenic differentiation.

Histone acetylation and deacetylation affect the patterns of gene expression in cellular differentiation, playing pivotal roles in tissue development and maintenance. For example, the intrinsic histone acetyltransferase activity of transcriptional coactivator p300 is especially required for the expression of myogenic regulatory factors including Myf5 and MyoD, and consequently for skeletal myogenesis. On the other hand, histone deacetylases (HDACs) remove the acetyl group from histones, which is critical for gene repression in stem cell fate transition. Through integrative omic analyses, we found that while some HDACs were differentially expressed at the early stage of skeletal myoblast differentiation, Hdac11 gene expression was significantly enhanced by nuclear receptor signaling. In addition, p300 and MyoD control Hdac11 expression in milieu of normal and signal-enhanced myoblast differentiation. Thus, HDAC11 may be essential to differential gene expression at the onset of myoblast differentiation.

Aberrant expression of CKS2 induced by ELK1 contributes to malignant progression of pancreatic cancer.

Cyclin-dependent kinase subunit 2 (CKS2) has been reported to promote various malignancies. This study investigated the functional role of CKS2 in pancreatic cancer (PC). An analysis of abnormally expressed genes and their prognostic value for PC was performed by using the Gene Expression Profiling Interactive Analysis (GEPIA) database and performing immunohistochemical staining on 64 samples of tumor tissue. CCK-8 assays, EdU staining, colony formation assays, flow cytometry, and a xenograft tumor model were used to analyze the biological function of CKS2 in PC. Our results revealed that CKS2 was expressed at significantly higher levels in PC tissues than in adjacent normal tissues, and a high level of CKS2 expression was associated with a poor prognosis for patients with PC. Moreover, functional assays revealed that CKS2 knockdown suppressed cell proliferation, induced cell cycle S phase, G2/M phase arrest, and apoptosis in vitro, and also reduced tumor growth in vivo. In addition, CKS2 knockdown increased the levels of Bax, caspase-3, P53, P21, and GADD45α expression, but decreased Bcl-2, Cyclin B1, CDK1, Cyclin A, and Cdc25C expression. CKS2 overexpression produced the opposite effects of CKS2 knockdown. Furthermore, we found that ELK1 protein regulated transcription of the CKS2 gene. In conclusion, our findings suggest that CKS2 expression is regulated by ELK1, which could possibly serve as prognostic indicator and therapeutic target for PC.

Walnut Kernel Oil and Defatted Extracts Enhance Mesenchymal Stem Cell Stemness and Delay Senescence.

Decreased stemness and increased cellular senescence impair the ability of mesenchymal stem cells (MSCs) to renew themselves, change into different cell types, and contribute to regenerative medicine. There is an urgent need to discover new compounds that can boost MSCs' stemness and delay senescence. Therefore, this study aimed to investigate the impact of walnut kernel oil (WKO) and defatted (WKD) extracts on bone marrow (BM)-MSC stemness and senescence. Premature senescence and inflammation were induced in BM-MSCs using H2O2 and LPS, respectively. Phytochemical constituents of WKO and WKD extracts were detected by HPLC. The stemness (proliferation and migration), senescence-related markers (p53, p21, SIRT1, and AMPK), oxidative stress/antioxidant markers, inflammatory cytokines, and cell cycle of BM-MSCs were measured by MTT assay, qPCR, ELISA, and flow cytometry. WKO and WKD extracts improved rat BM-MSC stemness, as evidenced by (1) increased cell viability, (2) decreased apoptosis (low levels of Bax and caspase3 and high levels of Bcl2), (3) upregulated MMP9 and downregulated TIMP1 expression, and (4) cell cycle arrest in the G0/G1 phase and declined cell number in the S and G2/M phases. Additionally, WKO and WKD extracts reduced rat BM-MSC senescence, as indicated by (1) decreased p53 and p21 expression, (2) upregulated expression and levels of SIRT1 and AMPK, (3) reduced levels of ROS and improved antioxidant activity (higher activity of CAT, SOD, and GPx and upregulated expression of NrF2 and HO-1), and (4) declined levels of TNFα, IL1β, and NF-κB. When compared to the WKO extract, the WKD extract had a greater impact on the induction of stemness and reduction of senescence of BM-MSCs due to its stronger antioxidant activity, which could be attributed to its higher levels of flavonoids and phenolic compounds, as detected by HPLC analysis. WKO and WKD extracts enhance rat BM-MSC stemness and protect them from senescence, suggesting their potential use as enhancers to increase MSCs' therapeutic efficacy.

RING box protein-1(RBX1), a key component of SCF E3 ligase, induced multiple myeloma cell drug-resistance though suppressing p27

ABSTRACT Multiple myeloma (MM) is a clonal disease of plasma cells that remains, for the most part, incurable despite the advent of several novel therapeutics. The elevated expression of p27 and its association with cell-cycle arrest is speculated to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents. In this study, we demonstrated that RBX1 silencing could inhibit MM cell growth and promote cell drug resistance. RBX1 directly interacted with and triggered the ubiquitination and degradation of p27, ultimately causing p27 reduction. Additionally, cell growth and apoptosis analysis indicated that the role of RBX1 in regulating myeloma cell proliferation and drug resistance resulted from p27 accumulation, which occurred in a Thr187 phosphorylation-dependent manner. Furthermore, the cell-cycle analysis demonstrated that RBX1 overexpression induced cells to enter the cell cycle (S-phase) and partially inhibited chemotherapeutic drugs-mediated cell cycle arrest. Notably, the forced expression of RBX1 also inhibited the cell adhesion-mediated elevation of p27 and induced the accumulation of adherent cells in apoptosis, especially the proteolytic cleavage of caspase-3. Additionally, RBX1 knockdown significantly inhibited myeloma development in SCID-Hu mice and in a human MM xenotransplant model. Overall, these in vitro and in vivo experiments indicated that the RBX1-p27 axis could be a central molecular mechanism by which RBX1 functions as a tumor promoter and stimulates cell growth in chemotherapeutic drugs treated MM cells.

CircRREB1 mediates lipid metabolism related senescent phenotypes in chondrocytes through FASN post-translational modifications

Osteoarthritis is a prevalent age-related disease characterized by dysregulation of extracellular matrix metabolism, lipid metabolism, and upregulation of senescence-associated secretory phenotypes. Herein, we clarify that CircRREB1 is highly expressed in secondary generation chondrocytes and its deficiency can alleviate FASN related senescent phenotypes and osteoarthritis progression. CircRREB1 impedes proteasome-mediated degradation of FASN by inhibiting acetylation-mediated ubiquitination. Meanwhile, CircRREB1 induces RanBP2-mediated SUMOylation of FASN and enhances its protein stability. CircRREB1-FASN axis inhibits FGF18 and FGFR3 mediated PI3K-AKT signal transduction, then increased p21 expression. Intra-articular injection of adenovirus–CircRreb1 reverses the protective effects in CircRreb1 deficiency mice. Further therapeutic interventions could have beneficial effects in identifying CircRREB1 as a potential prognostic and therapeutic target for age-related OA.