Cyclin-dependent Kinase Inhibitor P21 Research Articles

Regulatory effects of statins on Akt signaling for prevention of cancers

Statins, which are primarily used as lipid-lowering drugs, have been found to exhibit anti-tumor effects through modulating and interfering with various signaling pathways. In observational studies, statin use has been associated with a significant reduction in the progression of various cancers, including colon, lung, prostate, pancreas, and esophagus cancer, as well as melanoma and B and T cell lymphoma. The mevalonate pathway, which is affected by statins, plays a crucial role in activating Rho, Ras, and Rab proteins, thereby impacting the proliferation and apoptosis of tumor cells. Statins block this pathway, leading to the inhibition of isoprenoid units, which are critical for the activation of these key proteins, thereby affecting cancer cell behavior. Additionally, statins affect MAPK and Cdk2, which in turn reduce the expression of p21 and p27 cyclin-dependent kinase inhibitors. Akt signaling plays a crucial role in key cancer cell features like proliferation, invasion, and apoptosis by activating multiple effectors in downstream pathways such as FOXO, PTEN, NF-κB, GSK3β, and mTOR. The PI3K/Akt signaling is necessary for many events in the metastatic pathway and has been implicated in the resistance to cytostatic drugs. The Akt/PTEN axis is currently attracting great interest for its role in carcinogenesis. Statins have been shown to activate the purinergic receptor P2X7 and affect Akt signaling, which may have important anti-cancer effects. Hence, targeting Akt shows promise as an effective approach to cancer prevention and therapy. This review aims to provide a comprehensive discussion on the specific impact of statins through Akt signaling in different types of cancer.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Exercise Suppresses Head and Neck Squamous Cell Carcinoma Growth via Oncostatin M.

Major advances have been made in cancer treatment, but the prognosis for elderly cancer patients with sarcopenia and frailty remains poor. Myokines, which are thought to exert preventive effects against sarcopenia, have been reported to be associated with the prognosis of various cancers, but their effect on head and neck squamous cell carcinoma (HNSCC) is unknown. The aim of this study was to clarify the influence of exercise on the control of HNSCC and to examine the underlying mechanism involved. Mice were injected with HSC-3-M3 cells, a human cell line of highly metastatic and poorly differentiated tongue cancer, at the beginning of the study. Just prior to transplantation, blood was collected from the mice, and the levels of myokines were measured by ELISA. Oncostatin M (OSM), a selected myokine, was added to HSC-3-M3 cells, after which the cell proliferation ability, cell cycle, and protein expression were analyzed in vitro. Tumor cell viability was lower (control: 100%, exercise: 75%), tumors were smaller (control: 26.2 mm3, exercise: 6.4 mm3), and survival was longer in the exercise group than in the control group in vivo. OSM inhibited HSC-3-M3 cell proliferation in a concentration-dependent manner in vitro. The addition of OSM increased the proportion of cells in the G0/G1 phase, decreased the proportion of cells in the G2/M phase, and increased the expression of the CDK inhibitors p21 and p27. These results indicate that exercise may directly inhibit the proliferation of HNSCC cell lines via OSM.

Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21.

Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.

Skp2-Cyclin A Interaction Is Necessary for Mitotic Entry and Maintenance of Diploidy

Skp2, the substrate recognition component of the SCFSkp2 ubiquitin ligase, has been implicated in the targeted destruction of a number of key cell cycle regulators and the promotion of S-phase. One of its critical targets is the Cyclin dependent kinase (Cdk) inhibitor p27, and indeed the overexpression of Skp2 in a number of cancers is directly correlated with the premature degradation of p27. Skp2 was first identified as a protein that interacts with Cyclin A in transformed cells, but its role in this complex has remained unclear. In this paper, we demonstrate that Skp2 interacts with Cyclin A in Drosophila and is required to maintain Cyclin A levels and permit mitotic entry. Failure of mitotic entry in Skp2 mutant cells results in polyploidy. If these cells enter mitosis again they are unable to properly segregate their chromosomes, leading to checkpoint dependent cell cycle arrest or apoptosis. Thus, Skp2 is required for mitosis and for maintaining diploidy and genome stability.

Cyclohexene oxide CA, a derivative of zeylenone, exhibits anti-cancer activity in glioblastoma by inducing G0/G1 phase arrest through interference with EZH2.

Introduction: Due to its highly aggressiveness and malignancy, glioblastoma (GBM) urgently requires a safe and effective treatment strategy. Zeylenone, a natural polyoxygenated cyclohexenes compound isolated from Uvaria grandiflora, has exhibited potential biological activities in various human diseases, including tumors. Methods: We designed and synthesized a series of (+)-Zeylenone analogues and evaluated their anti-GBM roles through structural-activity analysis. Cell Counting Kit-8, TUNEL, transwell and flow cytometry were employed for investigating the anticancer effects of CA on GBM cells. Western blotting, molecular docking, qRT-PCR and ChIP assays were performed to reveal the underlying mechanisms by which CA regulates the GBM cell cycle. The nude mouse xenograft model, HE staining, immunohistochemistry and was used to evaluate the anticancer effect of CA in vivo. Results: We identified CA ((1R, 2R, 3S)-3-p-fluorobenzoyl-zeylenone) as having the lowest IC50 value in GBM cells. CA treatment significantly inhibited the malignant behaviors of GBM cells and induced G0/G1 phase arrest in vitro. Furthermore, we validated the molecular mechanism by which CA interferes with EZH2, attenuating the down-regulation of cyclin-dependent kinase inhibitors p27 and p16 by the PRC2 complex. By establishing orthotopic nude mice models, we further validated the inhibitory role of CA on tumorigenesis of GBM cells in vivo and its potential values to synergistically potentiate the anti-tumor effects of EZH2 inhibitors. Conclusion: Overall, this paper elucidated the anti-GBM effects and potential mechanisms of CA, and may provide a therapeutic drug candidate for GBM treatment.

Abstract LB_C06: Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic treatment options and a high rate of metastatic recurrence. TNBC is defined by lack of estrogen and progesterone receptor expression with non-amplified HER2, and accounts for 10-15% of all breast cancer cases. While pembrolizumab, sacituzumab govitecan and PARP inhibitors are beneficial for a subset of TNBC patients, chemotherapy including doxorubicin continues to be the primary treatment. Bocodepsin is a novel, orally bioavailable clinical stage Class 1-targeting HDAC inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate OKI-005 (in vitro) and bocodepsin (in vivo) in combination with doxorubicin (DOX) to overcome resistance. Procedures: TNBC cell lines were exposed to a no drug, DOX, OKI-005 or combination for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis was assessed following 48 hours of drug exposure by flow cytometry. Immunoblots were performed following 24 hours of drug treatment to evaluate mechanism of action. In vitro senescence-associated beta-galactosidase was performed after 6 days drug treatment. For in vivo studies, 5 million MDA-MB-231 cells were injected subcutaneously in athymic nude mice. Mice were treated with either no drug, 1.5 mg/kg DOX IP QW, 80 mg/kg bocodepsin PO QD, or the combination. Results: Bliss scores calculated from Synergy Finder+ using the CellTiter-Glo data showed synergy for the combination of OKI-005 and DOX in all TNBC cell lines. The combination resulted in statistically significant increased apoptosis measured by Annexin V compared to single agent drug treatment in CAL-51, MDA-MB-231 and CAL-120 cell lines. Immunoblot analysis demonstrated an increase in the pro-apoptotic protein BIM and decrease in anti-apoptotic protein BCL-XL with the combination. The cyclin dependent kinase inhibitor p21 was increased by OKI-005 independently of p53 and doxorubicin for the cell lines MDA-MB-231, Hs 578T, and CAL-120. The combination resulted in increased cleaved caspase-3 for all cell lines evaluated. Senescence-associated beta-galactosidase showed an increase in senescent cells for both DOX and OKI-005, which was eradicated in combination. DOX and bocodepsin were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to DOX (p=0.0016) and bocodepsin (p=0.0068). Conclusion: The addition of OKI-005 or bocodepsin to doxorubicin resulted in improved anti-proliferative and anti-cancer activity with evidence of increased apoptosis and decreased senescence. As a result, bocodepsin is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Bocodepsin has a favorable toxicity profile compared to other HDAC inhibitors and this work supports the potential clinical investigation of doxorubicin and bocodepsin in patients with metastatic TNBC. Citation Format: Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Evan D Dus, Stacey M Bagby, Adrian T A. Dominguez, Cameron A Binns, Jennifer R Diamond, Todd M Pitts. Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C06.

Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system.

Mast cell tumours (MCTs) arise in the dermis and subcutaneous tissues in animals and humans and are one of the most common neoplasms of the skin in dogs. Cannabinoid type 2 receptor (CB2R), cyclin-dependent kinase inhibitor (p21) and matrix metalloproteinase 1 (MMP-1) are potential targets for novel anti-tumour therapeutic strategies. This study evaluated by immunohistochemical means the reactivity of p21, MMP-1 and CB2R proteins in association with a three-tier grading system in cutaneous canine MCTs. Formalin-fixed, paraffin-embedded canine MCTs were processed for histochemical analysis and immunohistochemical staining using antibodies against p21, MMP-1 and CB2R. The results were analysed statistically. The strongest p21 immunolabelling was detected in grade 3 MCTs, while grade 1 tumours showed mild or no detectable p21 immunoreactivity (P-value < 0.001). Strong immunolabelling of MMP-1 was the most common in grade 1 tumours (P-value < 0.001) and CB2R was significantly less frequent in grade 3 tumours than in grade 1 (P-value < 0.001) and grade 2 (P-value < 0.001). High immunoreactivity of MMP-1 can be a marker of grade 1 MCTs in dogs, whereas p21 protein overexpression can be a marker of grade 3 canine MCTs. Strong CB2R immunoreactivity with simultaneous underexpression of p21 and high immunoreactivity of MMP-1 proteins may indicate that the use of cannabinoids in grade 1 MCTs in dogs is practicable.

Vasopressin Analog [V4Q5]dDAVP Exerts Cooperative Anticancer Effects in Combination With Low-Dose 5-Fluorouracil on Aggressive Colorectal Cancer Models.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Despite being an essential component of systemic chemotherapy for advanced CRC, 5-fluorouracil (5-FU) clinical use has severe limitations, such as high toxicity, low selectivity and drug resistance. [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) is a peptide vasopressin analog and a selective agonist of the arginine vasopressin type 2 membrane receptor (AVPR2), expressed in microvascular and tumor tissue. This synthetic compound has well-proven antitumor and antimetastatic activity in different tumor types, including metastatic CRC. The objective of this work was to assess the potential combinational benefits in preclinical CRC models after [V4Q5]dDAVP addition to 5-FU. Effects on cellular viability, cell cycle progression, apoptosis and molecular mechanisms associated to [V4Q5]dDAVP treatment in combination with 5-FU were evaluated in murine CT-26 and human COLO-205 cell lines. In vivo, impact of dual therapy was explored on CRC tumor growth and metastatic spread. In CRC cells, [V4Q5]dDAVP (1 µM) addition to sub-IC50 5-FU concentrations resulted in the enhancement of cytostatic effects induced by chemotherapy. Reduction of cell viability after combined treatment was associated with cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased gene expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the tumor suppressor p53 (TP53) in malignant cells, as assessed by flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. In vivo, intravenous administration of [V4Q5]dDAVP (0.3 µg/kg) in combination with safe low doses of 5-FU (50 or 80 mg/kg for CT-26 or COLO-205 tumor models, respectively) effectively abrogated CRC growth, reducing aggressiveness of primary lesions and increasing survival of tumor-bearing mice. In addition, concomitant administration of [V4Q5]dDAVP and 5-FU inhibited pulmonary metastasis formation by CT-26 cells in immunocompetent mice, especially reducing macrometastatic disease. [V4Q5]dDAVP seems to enhance the efficacy of 5-FU-based chemotherapy in CRC by modulating tumor progression, as well as metastatic dissemination, suggesting its potential role as a safe and cost-effective co-adjuvant agent for the management of advanced CRC.

Darolutamide Added to Docetaxel Augments Antitumor Effect in Models of Prostate Cancer through Cell Cycle Arrest at the G1-S Transition.

Resistance to taxane chemotherapy is frequently observed in metastatic prostate cancer. The androgen receptor (AR) is a major driver of prostate cancer and a key regulator of the G1-S cell-cycle checkpoint, promoting cancer cell proliferation by irreversible passage to the S-phase. We hypothesized that AR signaling inhibitor (ARSi) darolutamide in combination with docetaxel could augment antitumor effect by impeding the proliferation of taxane-resistant cancer cells. We monitored cell viability in organoids, tumor volume, and PSA secretion in patient-derived xenografts (PDX) and analyzed cell cycle and signaling pathway alterations. Combination treatment increased antitumor effect in androgen-sensitive, AR-positive prostate cancer organoids and PDXs. Equally beneficial effects of darolutamide added to docetaxel were observed in a castration-resistant model, progressive on docetaxel, enzalutamide, and cabazitaxel. In vitro studies showed that docetaxel treatment with simultaneous darolutamide resulted in a reduction of cells entering the S-phase in contrast to only docetaxel. Molecular analysis in the prostate cancer cell line LNCaP revealed an upregulation of cyclin-dependent kinase inhibitor p21, supporting blockade of S-phase entry and cell proliferation. Our results provide a preclinical support for combining taxanes and darolutamide as a multimodal treatment strategy in patients with metastatic prostate cancer progressive on ARSi and taxane chemotherapy.

Lipid-Mediated Modulation of DNA Damage Signaling As a Prognostic and Therapeutic Strategy Against Multiple Myeloma

Multiple myeloma (MM), characterized by the accumulation of tumor plasma cells in the bone marrow, is the second most common hematologic cancer. Occurrence of chemoresistance invariably leads patients to relapse and results in loss of clinical control over the disease. Cancer cells show a deregulation of different pathways, including lipid metabolism alterations and high genomic instability. Yet, no functional relationship between these two features has been identified. We have shown in a previous work that the metabolism of lipids impacts in how DNA damage is detected, signaled and repaired (Ovejero et al 2023 EMBO J). We reported that breaks in the DNA trigger the birth of lipid droplets (LD), a central lipid-storage organelle, and that this event is key for cells to tolerate this specific DNA damage. Given that MM cells are particularly dependent on DNA repair pathways for survival, we postulated that new therapeutic perspectives would emerge by targeting the formation of LD. Analysis of the LD formation gene expression profile of a cohort of newly diagnosed MM patients (UAMS-TT2 MM cohort, n = 345) allowed the establishment of a LD score comprising 13 genes associated with a significant prognostic value after multiple testing correction. The LD score gene expression-based risk score enabled the identification of MM patients with a poor outcome. The prognostic value of the LD score was validated in two other independent cohorts of newly diagnosed patients (HM cohort, n = 206; UAMS-TT3 MM cohort, n = 158) and in a cohort of patients at relapse treated with anti-CD38 MoAb (Mtp-Dara, n = 49). We subsequently studied the presence of LD in primary MM cells obtained from 34 patients. Importantly, we observed a significant positive correlation between the percentage of LD-positive cells and the value of the LD score (p = 0,02). This prompted us to investigate the therapeutic interest of interfering with LD formation to target MM cells. To this end, we treated 26 Human Myeloma Cell Lines (HMCLs) with drugs selected on an educated-guess basis and identified positive hits. These results were validated employing a shRNA strategy in selected HCMLs. Flow cytometry analyses revealed that this regime triggered a cell cycle halt, where cells accumulated in the G 1 phase at the expense of S and G 2. This was accompanied by a decrease in c-MYC expression, as well as an induction of cell proliferation arrest markers such as the G 1-checkpoint CDK inhibitors p21 and p27. Mechanistically, we observed a significant increase in Chk1 and Chk2 phosphorylations, two downstream events of DNA damage signaling despite that basal DNA damage levels remained unchanged. Together, these data convey the notion that limiting LD formation in MM cell lines boosts the detection of endogenously present DNA damage, thus enforcing a more robust DNA damage signaling which culminates in a drastic loss of proliferative capacity and viability. Of major importance, we validated the therapeutic interest of this strategy in primary MM cells from patients (n = 19). A significant higher toxicity in MM cells was found compared to normal cells from the microenvironment (tumoral: ****, p &amp;lt; 0.0001; normal: ***, p = 0,0006). Moreover, we tested the therapeutic interest of combining this approach with conventional treatments used in MM. Interestingly, we identified a synergistic effect when combined with melphalan, a DNA-alkylating agent used in MM treatment. This synergy was observed in HCMLs and in primary MM cells of patients (n = 7). Furthermore, significant synergistic effects were found in HMCLs by combining with the proteasome inhibitors bortezomib and carfilzomib. Altogether, these data unveil both the LD score and LD-bearing cell quantification as new prognostic factors in MM, and open new therapeutic perspectives to improve the treatment of MM patients by targeting LD metabolism.

Erratum: Down-Regulation of the Cyclin-Dependent kinase inhibitor p57 is mediated by Jab1/Csn5 in hepatocarcinogenesis.

Erratum: Down-Regulation of the Cyclin-Dependent kinase inhibitor p57 is mediated by Jab1/Csn5 in hepatocarcinogenesis.

Ebselen, Iron Uptake Inhibitor, Alleviates Iron Overload-Induced Senescence-Like Neuronal Cells SH-SY5Y via Suppressing the mTORC1 Signaling Pathway.

Increasing evidence highlights that excessive iron accumulation in the brain plays a vital role in neuronal senescence and is implicated in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, the chemical compounds that eliminate an iron overload may provide better protection against oxidative stress conditions that cause the accumulation of senescent cells during brain aging. Ebselen has been identified as a strongly useful compound in the research on redox biology mechanisms. We hypothesized that ebselen could alleviate an iron overload-induced oxidative stress and consequently reverses the senescence-like phenotypes in the neuronal cells. In the present study, SH-SY5Y cells were treated with ferric ammonium citrate (FAC) before ebselen, and the evaluation of the cellular iron homeostasis, the indicators of oxidative stress, and the onset of senescence phenotypes and mechanisms were carried out accordingly. Our findings showed that ebselen ameliorated the FAC-mediated iron overload by decreasing the expression of divalent metal transporter 1 (DMT1) and ferritin light chain (FT-L) proteins. In contrast, it increased the expression of ferroportin 1 (FPN1) protein and its correlation led to a decrease in the expression of the cytosolic labile iron pool (LIP). Furthermore, ebselen significantly reduced reactive oxygen species (ROS) and rescued the mitochondrial membrane potential (ΔΨm). Notably, ebselen restored the biomarkers of cellular senescence by reducing the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and senescence-associated secretory phenotypes (SASP). This also suppressed the expression of p53 protein targeting DNA damage response (DDR)/p21 cyclin-dependent kinase (CDK) inhibitor through a mTORC1 signaling pathway. Potentially, ebselen could be a therapeutic agent for treating brain aging and AD by mitigating iron accumulation and restoring senescence in SH-SY5Y cells.

Elevated CDKN1A (P21) mediates β-thalassemia erythroid apoptosis, but its loss does not improve β-thalassemic erythropoiesis

Abstractβ-thalassemias are common hemoglobinopathies due to mutations in the β-globin gene that lead to hemolytic anemias. Premature death of β-thalassemic erythroid precursors results in ineffective erythroid maturation, increased production of erythropoietin (EPO), expansion of erythroid progenitor compartment, extramedullary erythropoiesis, and splenomegaly. However, the molecular mechanism of erythroid apoptosis in β-thalassemia is not well understood. Using a mouse model of β-thalassemia (Hbbth3/+), we show that dysregulated expression of the FOXO3 transcription factor is implicated in β-thalassemia erythroid apoptosis. In Foxo3−/−/Hbbth3/+ mice, erythroid apoptosis is significantly reduced, whereas erythroid cell maturation, and red blood cell and hemoglobin production are substantially improved even with elevated reactive oxygen species in double-mutant erythroblasts. However, persistence of elevated reticulocytes and splenomegaly suggests that ineffective erythropoiesis is not resolved in Foxo3−/−/Hbbth3/+. We found the cell cycle inhibitor Cdkn1a (cyclin-dependent kinase inhibitor p21), a FOXO3 target gene, is markedly upregulated in both mouse and patient–derived β-thalassemic erythroid precursors. Double-mutant p21/Hbbth3/+ mice exhibited embryonic lethality with only a fraction of mice surviving to weaning. Notably, studies in adult mice displayed greatly reduced apoptosis and circulating Epo in erythroid compartments of surviving p21−/−/Hbbth3/+ mice relative to Hbbth3/+ mice, whereas ineffective erythroid cell maturation, extramedullary erythropoiesis, and splenomegaly were not modified. These combined results suggest that mechanisms that control β-thalassemic erythroid cell survival and differentiation are uncoupled from ineffective erythropoiesis and involve a molecular network including FOXO3 and P21. Overall, these studies provide a new framework for investigating ineffective erythropoiesis in β-thalassemia.

Pannexin 1 Modulates Angiogenic Activities of Human Endothelial Colony-Forming Cells Through IGF-1 Mechanism and Is a Marker of Senescence.

We examined the role of Panxs (pannexins) in human endothelial progenitor cell (EPC) senescence. Young and replication-induced senescent endothelial colony-forming cells (ECFCs) derived from human circulating EPCs were used to examine cellular activities and senescence-associated indicators after transfection of short interference RNA specific to Panx1 or lentivirus-mediated Panx1 overexpression. Hind limb ischemia mice were used as in vivo angiogenesis model. Protein and phospho-kinase arrays were used to determine underlying mechanisms. Panx1 was the predominant Panx isoform in human ECFCs and upregulated in both replication-induced senescent ECFCs and circulating EPCs from aged mice and humans. Cellular activities of the young ECFCs were enhanced by Panx1 downregulation but attenuated by its upregulation. In addition, reduction of Panx1 in the senescent ECFCs could rejuvenate cellular activities with reduced senescence-associated indicators, including senescence-associated β-galactosidase activity, p16INK4a (cyclin-dependent kinase inhibitor 2A), p21 (cyclin-dependent kinase inhibitor 1), acetyl-p53 (tumor protein P53), and phospho-histone H2A.X (histone family member X). In mouse ischemic hind limbs injected senescent ECFCs, blood perfusion ratio, salvaged limb outcome, and capillary density were all improved by Panx1 knockdown. IGF-1 (insulin-like growth factor 1) was significantly increased in the supernatant from senescent ECFCs after Panx1 knockdown. The enhanced activities and paracrine effects of Panx1 knockdown senescent ECFCs were completely inhibited by anti-IGF-1 antibodies. FAK (focal adhesion kinase), ERK (extracellular signal-regulated kinase), and STAT3 (signal transducer and activator of transcription 3) were activated in senescent ECFCs with Panx1 knockdown, in which the intracellular calcium level was reduced, and the activation was inhibited by supplemented calcium. The increased IGF-1 in Panx1-knockdown ECFCs was abrogated, respectively, by inhibitors of FAK (PF562271), ERK (U0126), and STAT3 (NSC74859) and supplemented calcium. Panx1 expression is upregulated in human ECFCs/EPCs with replication-induced senescence and during aging. Angiogenic potential of senescent ECFCs is improved by Panx1 reduction through increased IGF-1 production via activation of the FAK-ERK axis following calcium influx reduction. Our findings provide new strategies to evaluate EPC activities and rejuvenate senescent EPCs for therapeutic angiogenesis.

Histone H1.2 Represses the Transcription of the p16 Tumor Suppressor Gene.

CDK inhibitor p16 plays a pivotal role in the induction of cellular senescence and functions as a tumor suppressor. Here, we demonstrate that histone H1.2 is involved in p16 repression. Cells were transfected with siRNAs and subjected to quantitative reverse transcription-polymerase chain reaction, immunoblotting and chromatin immunoprecipitation (ChIP) assay. The decrease in H1.2 by oncogenic RAS was associated with increased levels of p16. Depletion of H1.2 selectively increased p16, but not alternative reading frame (ARF) mRNA. ChIP assay showed that H1.2 directly bound to the p16 promoter. Interestingly, silencing YB-1, a component of H1.2 complex, decreased the expression levels of H1.2, resulting in decreased binding of H1.2 on the p16 promoter. These results provide a model in which H1.2 is positively regulated by YB-1 and directly binds to and represses the transcription of p16.

P-391 Importance of oxygen tension in human ovarian tissue in vitro culture

Abstract Study question Is there any difference between 20% and 5% oxygen (O2) tension on the viability and quality of human follicles contained in cultured ovarian cortex? Summary answer 5% O2 yields higher follicle viability and quality than does 20% O2 tension after 6 days of in vitro culture (IVC). What is known already The primordial follicle (PMF) pool resides within the ovarian cortex, where in vivo O2 tension ranges between 2% and 8%. However, IVC is commonly performed at atmospheric O2 tension (20% O2), yielding low follicle survival rates. Elevated O2 tension or insufficient antioxidant protection can cause excessive reactive oxygen species (ROS) accumulation, leading to DNA lesions, such as oxidative stress damage and double-strand breaks (DSB). Study design, size, duration This prospective experimental study included frozen-thawed ovarian cortex from 6 adult patients (mean age: 28.5 years; age range: 26–31 years) undergoing laparoscopic surgery for non-ovarian diseases. Ovarian cortical fragments were cultured for 6 days at (i) 20% O2 with 5% CO2, and (ii) 5% O2 with 5% CO2. Non-cultured fragments served as controls. Participants/materials, setting, methods Cortical fragments were analyzed according to: hematoxylin and eosin (H&amp;E) staining for follicle count and classification; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine (8-OHdG) and gamma-H2AX (γH2AX) immunolabeling to detect oxidative stress damage and DNA DSBs; and β-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore gene expression of superoxide dismutase 2 (SOD2) from the antioxidant defense system, and cyclin-dependent kinase inhibitors (p21 and p16) as senescence-related genes. Main results and the role of chance Apoptosis (p = 0.002) and follicle senescence (p = 0.03) rates were significantly lower in the 5% O2 than the 20% O2 group. Moreover, granulosa cells (GCs) in follicles in the 20% O2 group exhibited significantly (p &amp;lt; 0.001) higher oxidative stress damage rates than in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (p = 0.001) in the 20% O2 than the 5% O2 group. SOD2 values were significantly greater in the 5% O2 group compared to the 20% O2 group (p = 0.04) and the non-cultured group (p = 0.002). Expression of p21 was significantly increased in 20% O2 (p = 0.03) and 5% O2 (p = 0.008) compared to no culture. Moreover, the 20% O2 group showed significantly greater p16 expression (p = 0.04) than the non-cultured group, while no significant variation was observed between 5% O2 and no culture. Limitations, reasons for caution This study focuses on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue. The impact of O2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. Wider implications of the findings Our findings suggest that 5% O2 tension culture is a promising step towards potentially solving the problem of poor follicle viability after IVC. Trial registration number N/A

Importance of oxygen tension in human ovarian tissue in vitro culture.

Is there any difference between 20% and 5% oxygen (O2) tension in vitro culture (IVC) on the viability and quality of human follicles contained in cultured ovarian cortex? An O2 tension of 5% yields higher follicle viability and quality than does 20% O2 tension after 6 days of IVC. The primordial follicle (PMF) pool resides within the ovarian cortex, where the in vivo O2 tension ranges between 2% and 8%. Some studies suggest that lowering O2 tension to physiological levels may improve in vitro follicle quality rates. This prospective experimental study included frozen-thawed ovarian cortex from six adult patients (mean age: 28.5 years; age range: 26-31 years) who were undergoing laparoscopic surgery for non-ovarian diseases. Ovarian cortical fragments were cultured for 6 days at (i) 20% O2 with 5% CO2 and (ii) 5% O2 with 5% CO2. Non-cultured fragments served as controls. Cortical fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; Ki67 staining to evaluate PMF proliferation; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine and gamma-H2AX (γH2AX) immunolabeling to detect oxidative stress damage and DNA double-strand breaks (DSBs) in oocytes and granulosa cells (GCs); and β-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore the gene expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase 4 (GPX4) from the antioxidant defense system and cyclin-dependent kinase inhibitors (p21 and p16) as tissue senescence-related genes. Apoptosis (P = 0.002) and follicle senescence (P < 0.001) rates were significantly lower in the 5% O2 group than in the 20% O2 group. Moreover, GCs in follicles in the 20% O2 group exhibited significantly (P < 0.001) higher oxidative stress damage rates than those in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (P = 0.001) in the 20% O2 group than in the 5% O2 group. SOD2 expression was significantly greater in the 5% O2 group compared to the 20% O2 group (P = 0.04) and the non-cultured group (P = 0.002). Expression of p21 was significantly increased in both the 20% O2 (P = 0.03) and 5% O2 (P = 0.008) groups compared to the non-cultured group. Moreover, the 20% O2 group showed significantly greater p16 expression (P = 0.04) than the non-cultured group, while no significant variation was observed between the 5% O2 and no culture groups. N/A. This study focuses on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue. The impact of O2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. Our findings suggest that 5% O2 tension culture is a promising step toward potentially solving the problem of poor follicle viability after IVC. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0064.22, CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.). The authors have nothing to disclose.

Expression and significance of cyclinD1, cyclin-dependent kinase4 and cyclin-dependent kinase inhibitor P27 in patients with non-neoplastic epithelial disorders of the vulva.

Non-neoplastic epithelial disorders of the vulva (NNEDV) are prevalent and refractory gynecological diseases. However, the underlying pathogenesis of these diseases remain unclear. The present study aimed to investigate the expression and significance of cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase inhibitor P27 (P27) in patients with NNEDV and provide a reference for clinical diagnosis and treatment. Normal vulvar skin samples from patients with perineum repair (control group, n=20) and skin samples from the vulvar lesions of patients with NNEDV (NNEDV group, n=36) were collected. Expression levels of cyclin D1, CDK4 and P27 were assessed in the samples using immunohistochemistry. The expression of each protein was evaluated based on the mean optical density (MOD). The MODs of cyclin D1 and CDK4 were significantly higher in samples of the three pathological types of NNEDV, namely squamous hyperplasia (SH), lichen sclerosus (LS) and mixed SH and LS lesions, compared with those of the control group. The MOD of P27 was lower in samples of the three pathological types of NNEDV than in the control group, although the difference was not statistically significant. No significant differences in the MOD of cyclin D1, CDK4 and P27 were detected among the three pathological types of NNEDV. The ratios of the MOD of cyclin D1 and CDK4 in the prickle cell layer to those in the basal cell layer were significantly higher in the NNEDV group than in the control group. However, the ratio of the MOD of P27 in the prickle cell layer to that in the basal cell layer exhibited no significant difference between the NNEDV and control groups. NNEDV has the potential for malignant transformation. The occurrence and development of NNEDV may be associated with the acceleration of cell proliferation, in which cyclin D1, CDK4 and P27 contribute to regulation of the cell cycle. Therefore, cyclin D1, CDK4 and P27 may be potential targets in the development of new clinical therapeutic drugs for patients with NNEDV.

Connexin 37 sequestering of activated-ERK in the cytoplasm promotes p27-mediated endothelial cell cycle arrest.

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.