Essential role of p21Waf1/Cip1 in the modulation of post-traumatic hippocampal Neural Stem Cells response.

BACKGROUND: Traumatic Brain Injury (TBI) represents one of the main causes of brain damage in young people and the elderly population with a very high rate of psycho-physical disability and death. TBI is characterized by extensive cell death, tissue damage and neuro-inflammation with a symptomatology that varies depending on the severity of the trauma from memory loss to a state of irreversible coma and death. Recently, preclinical studies on mouse models have demonstrated that the post-traumatic adult Neural Stem/Progenitor cells response could represent an excellent model to shed light on the neuro-reparative role of adult neurogenesis following damage. The cyclin-dependent kinase inhibitor p21Waf1/Cip1 plays a pivotal role in modulating the quiescence/activation balance of adult Neural Stem Cells (aNSCs) and in restraining the proliferation progression of progenitor cells. Based on these considerations, the aim of this work is to evaluate how the conditional ablation of p21Waf1/Cip1 in the aNSCS can alter the adult hippocampal neurogenesis in physiological and post-traumatic conditions. METHODS: We designed a novel conditional p21Waf1/Cip1 knock-out mouse model, in which the deletion of p21Waf1/Cip1 (referred as p21) is temporally controlled and occurs in Nestin-positive aNSCs, following administration of Tamoxifen. This mouse model (referred as p21 cKO mice) was subjected to Controlled Cortical Impact to analyze how the deletion of p21 could influence the post-traumatic neurogenic response within the hippocampal niche. RESULTS: The data demonstrates that the conditional deletion of p21 in the aNSCs induces a strong increase in activation of aNSCs as well as proliferation and differentiation of neural progenitors in the adult dentate gyrus of the hippocampus, resulting in an enhancement of neurogenesis and the hippocampal-dependent working memory. However, following traumatic brain injury, the increased neurogenic response of aNSCs in p21 cKO mice leads to a fast depletion of the aNSCs pool, followed by declined neurogenesis and impaired hippocampal functionality. CONCLUSIONS: These data demonstrate for the first time a fundamental role of p21 in modulating the post-traumatic hippocampal neurogenic response, by the regulation of the proliferative and differentiative steps of aNSCs/progenitor populations after brain damage.

Antitumor Effect of Apcin on Endometrial Carcinoma via p21-Mediated Cell Cycle Arrest and Apoptosis.

OBJECTIVE: Endometrial carcinoma (EC) is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates. This underscores the critical need for novel therapeutic targets. One such potential target is cell division cycle 20 (CDC20), which has been implicated in oncogenesis. This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved. METHODS: The effects of Apcin on EC cell proliferation, apoptosis, and the cell cycle were evaluated using CCK8 assays and flow cytometry. RNA sequencing (RNA-seq) was subsequently conducted to explore the underlying molecular mechanism, and Western blotting and coimmunoprecipitation were subsequently performed to validate the results. Animal studies were performed to evaluate the antitumor effects in vivo. Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC. RESULTS: Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells, resulting in cell cycle arrest. Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin. Notably, Apcin treatment led to the upregulation of the cell cycle regulator p21, which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells. In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth. Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue, and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval. CONCLUSION: CDC20 is a novel molecular target in EC, and Apcin could be developed as a candidate antitumor drug for EC treatment.

The protective effects of orexin-A in alleviating cell senescence against interleukin-1ß (IL-1ß) in chondrocytes.

Osteoarthritis (OA) is one of the most important causes of global disability, and dysfunction of chondrocytes is an important risk factor. The treatment of OA is still a challenge. Orexin-A is a hypothalamic peptide, and its effects in OA are unknown. In this study, we found that exposure to interleukin-1ß (IL-1ß) reduced the expression of orexin-2R, the receptor of orexin-A in TC-28a2 chondrocytes. Importantly, the senescence-associated ß-galactosidase (SA-ß-gal) staining assay demonstrated that orexin-A treatment ameliorates IL-1ß-induced cellular senescence. Importantly, the presence of IL-1ß significantly reduced the telomerase activity of TC-28a2 chondrocytes, which was rescued by orexin-A. We also found that orexin-A prevented IL-1ß-induced increase in the levels of Acetyl-p53 and the expression of p21. It is shown that orexin-A mitigates IL-1ß-induced reduction of sirtuin 3 (SIRT3). Silencing of SIRT3 abolished the protective effects of orexin-A against IL-1ß-induced cellular senescence. These results imply that orexin-A might serve as a promising therapeutic agent for OA.

Inhibition of endogenous hydrogen sulfide production reduces activation of hepatic stellate cells via the induction of cellular senescence.

In chronic liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into activated myofibroblast-like cells and produce large amounts of extracellular matrix components, e.g. collagen type 1. Cellular senescence is characterized by irreversible cell-cycle arrest, arrested cell proliferation and the acquisition of the senescence-associated secretory phenotype (SASP) and reversal of HSCs activation. Previous studies reported that H2S prevents induction of senescence via its antioxidant activity. We hypothesized that inhibition of endogenous H2S production induces cellular senescence and reduces activation of HSCs. Rat HSCs were isolated and culture-activated for 7 days. After activation, HSCs treated with H2S slow-releasing donor GYY4137 and/or DL-propargylglycine (DL-PAG), an inhibitor of the H2S-producing enzyme cystathionine γ-lyase (CTH), as well as the PI3K inhibitor LY294002. In our result, CTH expression was significantly increased in fully activated HSCs compared to quiescent HSCs and was also observed in activated stellate cells in a in vivo model of cirrhosis. Inhibition of CTH reduced proliferation and expression of fibrotic markers Col1a1 and Acta2 in HSCs. Concomitantly, DL-PAG increased the cell-cycle arrest markers Cdkn1a (p21), p53 and the SASP marker Il6. Additionally, the number of ß-galactosidase positive senescent HSCs was increased. GYY4137 partially restored the proliferation of senescent HSCs and attenuated the DL-PAG-induced senescent phenotype. Inhibition of PI3K partially reversed the senescence phenotype of HSCs induced by DL-PAG. Inhibition of endogenous H2S production reduces HSCs activation via induction of cellular senescence in a PI3K-Akt dependent manner. Our results show that cell-specific inhibition of H2S could be a novel target for anti-fibrotic therapy via induced cell senescence.

Loss of CFTR Reverses Senescence Hallmarks in SARS-CoV-2 Infected Bronchial Epithelial Cells.

SARS-CoV-2 infection has been recently shown to induce cellular senescence in vivo. A senescence-like phenotype has been reported in cystic fibrosis (CF) cellular models. Since the previously published data highlighted a low impact of SARS-CoV-2 on CFTR-defective cells, here we aimed to investigate the senescence hallmarks in SARS-CoV-2 infection in the context of a loss of CFTR expression/function. We infected WT and CFTR KO 16HBE14o-cells with SARS-CoV-2 and analyzed both the p21 and Ki67 expression using immunohistochemistry and viral and p21 gene expression using real-time PCR. Prior to SARS-CoV-2 infection, CFTR KO cells displayed a higher p21 and lower Ki67 expression than WT cells. We detected lipid accumulation in CFTR KO cells, identified as lipolysosomes and residual bodies at the subcellular/ultrastructure level. After SARS-CoV-2 infection, the situation reversed, with low p21 and high Ki67 expression, as well as reduced viral gene expression in CFTR KO cells. Thus, the activation of cellular senescence pathways in CFTR-defective cells was reversed by SARS-CoV-2 infection while they were activated in CFTR WT cells. These data uncover a different response of CF and non-CF bronchial epithelial cell models to SARS-CoV-2 infection and contribute to uncovering the molecular mechanisms behind the reduced clinical impact of COVID-19 in CF patients.

PCNA molecular recognition of different PIP motifs: Role of Tyr211 phosphorylation.

The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimeric protein that embraces the DNA duplex. Many proteins bind PCNA through a conserved sequence known as the PCNA interacting protein motif (PIP). PCNA is further regulated by different post-translational modifications. Phosphorylation at residue Y211 facilitates unlocking stalled replication forks to bypass DNA damage repair processes but increasing nucleotide misincorporation. We explore here how phosphorylation at Y211 affects PCNA recognition of the canonical PIP sequences of the regulatory proteins p21 and p15, which bind with nM and µM affinity, respectively. For that purpose, we have prepared PCNA with p-carboxymethyl-L-phenylalanine (pCMF, a mimetic of phosphorylated tyrosine) at position 211. We have also characterized PCNA binding to the non-canonical PIP sequence of the catalytic subunit of DNA polymerase δ (p125), and to the canonical PIP sequence of the enzyme ubiquitin specific peptidase 29 (USP29) which deubiquitinates PCNA. Our results show that Tyr211 phosphorylation has little effect on the molecular recognition of p21 and p15, and that the PIP sequences of p125 and USP29 bind to the same site on PCNA as other PIP sequences, but with very low affinity.

Enzyme-independent role of EZH2 in regulating cell cycle progression via the SKP2-KIP/CIP pathway.

While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.

Genome-wide in vivo CRISPR screen identifies TGFß3 as actionable biomarker of palbociclib resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFß3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFß3 treatment can overcome this. This study defines TGFß3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFß3 to propose a new combinatorial treatment for TNBC.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

Resveratrol nanoparticles induce apoptosis in oral cancer stem cells by disrupting the interaction between ß-catenin and GLI-1 through p53-independent activation of p21.

Cancer stem cells (CSCs) are mainly responsible for tumorigenesis, chemoresistance, and cancer recurrence. CSCs growth and progression are regulated by multiple signaling cascades including Wnt/ß-catenin and Hh/GLI-1, which acts independently or via crosstalk. Targeting the crosstalk of signaling pathways would be an effective approach to control the CSC population. Both Wnt/ß-catenin and Hh/GLI-1 signaling cascades are known to be regulated by p53/p21-dependent mechanism. However, it is interesting to delineate whether p21 can induce apoptosis in a p53-independent manner. Therefore, utilizing various subtypes of oral CSCs (SCC9-PEMT p53+/+p21+/+, SCC9-PEMT p53-/-p21+/+, SCC9-PEMT p53+/+p21-/- and SCC9-PEMT p53-/-p21-/-), we have examined the distinct roles of p53 and p21 in Resveratrol nanoparticle (Res-Nano)-mediated apoptosis. It is interesting to see that, besides the p53/p21-mediated mechanism, Res-Nano exposure also significantly induced apoptosis in oral CSCs through a p53-independent activation of p21. Additionally, Res-Nano-induced p21-activation deregulated the ß-catenin-GLI-1 complex and consequently reduced the TCF/LEF and GLI-1 reporter activities. In agreement with in vitro data, similar experimental results were obtained in in vivo mice xenograft model.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ versus p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Exploring CDKN1A Upregulation Mechanisms: Insights into Cell Cycle Arrest Induced by NC2603 Curcumin Analog in MCF-7 Breast Cancer Cells.

Breast cancer stands out as one of the most prevalent malignancies worldwide, necessitating a nuanced understanding of its molecular underpinnings for effective treatment. Hormone receptors in breast cancer cells substantially influence treatment strategies, dictating therapeutic approaches in clinical settings, serving as a guide for drug development, and aiming to enhance treatment specificity and efficacy. Natural compounds, such as curcumin, offer a diverse array of chemical structures with promising therapeutic potential. Despite curcumin's benefits, challenges like poor solubility and rapid metabolism have spurred the exploration of analogs. Here, we evaluated the efficacy of the curcumin analog NC2603 to induce cell cycle arrest in MCF-7 breast cancer cells and explored its molecular mechanisms. Our findings reveal potent inhibition of cell viability (IC50 = 5.6 µM) and greater specificity than doxorubicin toward MCF-7 vs. non-cancer HaCaT cells. Transcriptome analysis identified 12,055 modulated genes, most notably upregulation of GADD45A and downregulation of ESR1, implicating CDKN1A-mediated regulation of proliferation and cell cycle genes. We hypothesize that the curcumin analog by inducing GADD45A expression and repressing ESR1, triggers the expression of CDKN1A, which in turn downregulates the expression of many important genes of proliferation and the cell cycle. These insights advance our understanding of curcumin analogs' therapeutic potential, highlighting not just their role in treatment, but also the molecular pathways involved in their activity toward breast cancer cells.

VBP1 promotes tumor proliferation as a part of the hypoxia-related signature in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor in East Asia. Hypoxia, a hallmark of solid tumors, significantly alters redox homeostasis inside tumor microenvironment. This alteration drives tumor proliferation, invasion, and metastasis, leading to poor prognostic outcomes. However, the role of hypoxia-related genes in ESCC remains poorly understood. We employed RNA sequencing to identify differentially expressed genes in ESCC. Clinical data, transcriptome profiles, and a hypoxia-related gene set were extracted from open-source databases. A prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression, which was then validated through Cox regression analysis. Within this prognostic model, we pinpointed and investigated a key hypoxia-related gene affecting prognosis. The gene's expression was validated using real-time PCR and immunohistochemistry in both esophageal carcinoma and normal tissues. Tumor proliferation was examined through in vitro and in vivo assays, including the Cell Counting Kit-8, EdU, colony formation, and subcutaneous tumor models. A robust four-gene prognostic model (VBP1, BGN, CDKN1A, and PPFIA1) was successfully constructed and validated. Among these, VBP1 emerged as a key gene, exhibiting high expression levels that correlated with poor prognosis in ESCC. Functional experiments confirmed that VBP1 significantly accelerated tumor proliferation both in vitro and in vivo. VBP1 is identified as a pivotal gene within the hypoxia-related prognostic signature, and it significantly promotes tumor proliferation in ESCC.

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-ß-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence.

Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.

Homogentisic acid metabolism inhibits papillary thyroid carcinoma proliferation through ROS and p21-induced cell cycle arrest.

Thyroid cancer is one of the most common primary endocrine malignancies worldwide, and papillary thyroid carcinoma (PTC) is the predominant histological type observed therein. Although PTC has been studied extensively, our understanding of the altered metabolism and metabolic profile of PTC tumors is limited. We identified that the content of metabolite homogentisic acid (HGA) in PTC tissues was lower than that in adjacent non-cancerous tissues. We evaluated the potential of HGA as a novel molecular marker in the diagnosis of PTC tumors, as well as its ability to indicate the degree of malignancy. Studies have further shown that HGA contributes to reactive oxygen species (ROS) associated oxidative stress, leading to toxicity and inhibition of proliferation. In addition, HGA caused an increase in p21 expression levels in PTC cells and induced G1 arrest. Moreover, we found that the low HGA content in PTC tumors was due to the low expression levels of tyrosine aminotransferase (TAT) and p-hydroxyphenylpyruvate hydroxylase (HPD), which catalyze the conversion of tyrosine to HGA. The low expression levels of TAT and HPD are strongly associated with a higher probability of PTC tumor invasion and metastasis. Our study demonstrates that HGA could be used to diagnose PTC and provides mechanisms linking altered HGA levels to the biological behavior of PTC tumors.

Ze-Qi-Tang formula inhibits MDSCs glycolysis through the down-regulation of p21/Hif1α/Glut1 signal in psoriatic-like mice.

BACKGROUND: Psoriasis is a chronic immune-mediated inflammatory skin disease that affects the quality of life and mental health of approximately 150 million people worldwide. Ze-Qi-Tang (ZQT) is a classic compound used in China for lung disease; however, its mechanism of action in psoriasis remains unclear. This study aimed to investigate the therapeutic effect of the ZQT formula on psoriasis and explore the underlying molecular mechanisms. METHODS: Peripheral blood samples were collected from patients with psoriasis and healthy individuals. Flow cytometry was used to detect changes in the proportions of myeloid-derived suppressor cells (MDSCs) and other immune cells. Psoriasis was induced in mice by the daily application of imiquimod. ZQT was administered separately or in combination with anti-Gr1 antibody to deplete MDSC. The glycolysis levels of the MDSCs were detected using a Seahorse analyzer. The p21/Hif1α/Glut1 pathway was identified and validated by mRNA sequence, RT-qPCR, WB, IF, and the application of p21 inhibitor UC2288. RESULTS: The number of MDSCs was significantly increased in patients with psoriasis, with the increased expression of p21, Hif1α, and Glut1 in MDSCs. ZQT significantly alleviated psoriasis-like skin lesions in mice. ZQT formula significantly reduced the number of MDSCs in psoriatic-like mice and enhanced their suppressive capacity for T cells. The efficacy of ZQT in alleviating psoriatic dermatitis is compromised by MDSC depletion. ZQT decreased the expressions of p21, Hif1α, and Glut1-induced glycolysis in MDSCs, thereby inhibiting Th17 cell differentiation. CONCLUSION: These suggest that ZQT alleviates IMQ-induced psoriatic dermatitis, by inhibiting p21/Hif1α/Glut1-induced glycolysis in MDSCs.

Ketogenic diet induces p53-dependent cellular senescence in multiple organs.

A ketogenic diet (KD) is a high-fat, low-carbohydrate diet that leads to the generation of ketones. While KDs improve certain health conditions and are popular for weight loss, detrimental effects have also been reported. Here, we show mice on two different KDs and, at different ages, induce cellular senescence in multiple organs, including the heart and kidney. This effect is mediated through adenosine monophosphate-activated protein kinase (AMPK) and inactivation of mouse double minute 2 (MDM2) by caspase-2, leading to p53 accumulation and p21 induction. This was established using p53 and caspase-2 knockout mice and inhibitors to AMPK, p21, and caspase-2. In addition, senescence-associated secretory phenotype biomarkers were elevated in serum from mice on a KD and in plasma samples from patients on a KD clinical trial. Cellular senescence was eliminated by a senolytic and prevented by an intermittent KD. These results have important clinical implications, suggesting that the effects of a KD are contextual and likely require individual optimization.

Cell Cycle Checkpoints p16 and p21-Strong Predictors of Clinicopathologic Outcomes in High-Grade Osteosarcoma.

PURPOSE: In this study, we used a series of immunohistochemical measurements of 2 cell cycle regulators, p16 and p21, to evaluate their prognostic value, separately and in combination, for the disease outcomes. METHOD: A total of 101 patients with high-grade osteosarcoma were included in this study. Clinicopathologic data were collected, and immunohistochemistry for p16 and p21 was performed and interpreted by 3 independent pathologists. Statistical analysis was performed to assess the strength of each of these markers relative to disease outcome. RESULTS: Our results indicate that more than 90% expression (high) of p16 by immunohistochemistry on the initial biopsy has a strong predictive value for good histologic response to chemotherapy. The patients are also more likely to survive the past 5 years and less likely to develop metastasis than patients with less than 90% p16 (low) expression. The results for p21, on the other hand, show a unique pattern of relationship to the clinicopathologic outcomes of the disease. Patients with less than 1% (low) or more than 50% (high) expression of p21 by immunohistochemistry show a higher chance of metastasis, poor necrotic response to chemotherapy, and an overall decreased survival rate when compared with p21 expression between 1% and 50% (moderate). Our results also showed that the expression of p16 and combined p16 and p21 demonstrates a stronger predictive relationship to 5-year survival than tumor histologic necrosis and p21 alone. DISCUSSION: The results of this study, once proven to be reproducible by a larger number of patients, will be valuable in the initial assessment and risk stratification of the patients for treatment and possibly the clinical trials.