Cells In G1 Phase Research Articles

Design, Synthesis, and Preclinical Activity in Ovarian Cancer Models of New Phosphanegold(I)-N-heterocyclic Carbene Complexes.

A new series of seven gold(I) complexes (1-7) containing 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene (IPr) and phosphane ligands (L1-L7) were synthesized and evaluated for antitumor activity in ovarian cancer (OvCa) models. The synthesized complexes were characterized by IR, mass spectrometry and NMR spectroscopy, and complex 6 was characterized by XRD crystallography. The antiproliferative effect of the new complexes (1-7) was found to be higher than cisplatin and auranofin in OvCa cells sensitive and resistant to cisplatin. The anticancer activity of the most active complex 6 was investigated using OvCa in vitro models, including three-dimensional (3D) multicellular tumor spheroids and in vivo tumor xenografts. Both cisplatin and auranofin were used for comparative purposes. Complex 6 induced apoptosis, mitochondrial reactive oxygen species, and DNA damage; caused a G1 phase cell cycle arrest, inhibited proteasome activity, and cell migration; modified actin polymerization; and significantly inhibited OvCa murine xenografts. These promising results suggest further preclinical testing of these complexes for future applications.

CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.

Dynamic biological characteristics of human bone marrow hematopoietic stem cell senescence

Hematopoietic stem cells show biological manifestations of aging, diminished hematopoietic function and abnormal differentiation, which can lead to leukemia. It is therefore important to explore the mechanism underlying hematopoietic stem cell aging to develop strategies for delaying the process. Our evaluations revealed that the number of bone marrow hematopoietic cells (BMHCs) started to decrease significantly after 45 years of age, and the number of senescent BMHCs, as determined by senescence-associated beta-galactosidase staining, gradually increased with age. In addition, BMHCs from individuals over 45 years of age presented with notably reduced proliferative capacity, increased G1-phase cell cycle arrest, and significantly decreased generation of mixed colony forming units, which suggests that BMHCs enter senescence during middle age. Furthermore, we observed significantly lower antioxidant capacity and a significant increase in oxidative damage products, a gradual increase in the expression of senescence-associated proteins and genes, and a gradual decrease in the expression of cell cycle related proteins in BMHCs after middle age. Taken together, these findings offer both a theoretical and experimental basis for better understanding of the senescence progression of BMHCs and the optimal timing for anti-senescence drug interventions in clinical practice.

PH-sensitive nanomedicine of novel tubulin polymerization inhibitor for lung metastatic melanoma.

Microtubule binding agents such as paclitaxel and vincristine have activity in metastatic melanoma. However, even responsive tumors develop resistance, highlighting the need to investigate new drug molecules. Here, we showed that a new compound, CH-2-102, developed by our group, has high anti-tumor efficacy in human and murine melanoma cells. We confirmed that CH-2-102 robustly suppresses the microtubule polymerization process by directly interacting with the colchicine binding site. Our results unveil that CH-2-102 suppresses microtubule polymerization and subsequently induces G2 phase cell arrest as one of the possible mechanisms. Notably, CH-2-102 maintains its efficacy even in the paclitaxel resistance melanoma cells due to different binding sites and a non-Pgp substrate. We developed a pH-responsive drug-polymer Schiff bases linker for high drug loading into nanoparticles (NPs). Our CH-2-102 conjugated NPs induced tumor regression more effectively than Abraxane® (Nab-paclitaxel, N-PTX), free drug, and non-sensitive NPs in B16-F10 cell-derived lung metastasis mouse model. Furthermore, our results suggest that the formulation has a high impact on the in vivo efficacy of the drug and warrants further investigation in other cancers, particularly taxane resistant. In conclusion, the microtubule polymerization inhibitor CH-2-102 conjugated pH-responsive NPs induce tumor regression in lung metastasis melanoma mice, suggesting it may be an effective strategy for treating metastatic melanoma.

Glabridin inhibits urothelial bladder carcinoma cell growth in vitro and in vivo by inducing cell apoptosis and cell cycle arrest.

Glabridin (GLA) has a variety of biological activities and therapeutic effects in cancers. Whereas the effect of GLA on urothelial bladder carcinoma (UBC) cells and its underlying mechanisms remain unknown. The study revealed the effect of GLA on UBC and the potential mechanism of inducing cell apoptosis in vivo and in vitro. After treated with different concentrations of GLA, the cell activity decreased in a time- and dose-dependent manner. The IC50 values of BIU-87 and EJ cells at 48 h were 6.02 μg/ml (18.6 μm) and 4.36 μg/ml(13.4μm), respectively. Additionally, GLA-induced apoptosis and cycle arrest of BIU-87 and EJ cells in G2 phase. Furthermore, wound healing experiments showed that GLA significantly reduced the migration activities of BIU-87 and EJ cells. Mechanically, GLA obviously increased the expression of BIM, BAK1, and CYCS in both mRNA and protein levels, which led to the activation of the endogenous apoptotic pathway. Finally, GLA remarkably inhibited the growth of UBC tumors in vivo. In summary, GLA inhibited UBC cells growth in vitro and in vivo by inducing cell apoptosis and cell cycle arrest, highlighting that GLA could be utilized as a component to design a novel anti-UBC drug.

MED16 Promotes Tumour Progression and Tamoxifen Sensitivity by Modulating Autophagy through the mTOR Signalling Pathway in ER-Positive Breast Cancer.

Recent studies have shown that the mediator complex (MED) plays a vital role in tumorigenesis and development, but the role of MED16 (mediator complex subunit 16) in breast cancer (BC) is not clear. Increasing evidence has shown that the mTOR pathway is important for tumour progression and therapy. In this study, we demonstrated that the mTOR signalling pathway is regulated by the expression level of MED16 in ER+ breast cancer. With the analysis of bioinformatics data and clinical specimens, we revealed an elevated expression of MED16 in luminal subtype tumours. We found that MED16 knockdown significantly inhibited cell proliferation and promoted G1 phase cell cycle arrest in ER+ BC cell lines. Downregulation of MED16 markedly reduced the sensitivity of ER+ BC cells to tamoxifen and increased the stemness and autophagy of ER+ BC cells. Bioinformatic analysis of similar genes to MED16 were mainly enriched in autophagy, endocrine therapy and mTOR signalling pathways, and the inhibition of mTOR-mediated autophagy restored sensitivity to tamoxifen by MED16 downregulation in ER+ BC cells. These results suggest an important role of MED16 in the regulation of tamoxifen sensitivity in ER+ BC cells, crosstalk between the mTOR signalling pathway-induced autophagy, and together, with the exploration of tamoxifen resistance, may indicate a new therapy option for endocrine therapy-resistant patients.

Single-nucleus RNA Sequencing reveals the mechanism of cigarette smoke exposure on diminished ovarian reserve in mice

The systematic toxicological mechanism of cigarette smoke (CS) on ovarian reserve has not been extensively investigated. Female 8-week-old C57BL/6 mice at peak fertility were exposed to CS or indoor air only for 30 days (100 mice per group) and the effects of CS on ovarian reserve were assessed using Single-Nucleus RNA Sequencing (snRNA-seq). In addition, further biochemical experiments, including immunohistochemical staining, ELISA, immunofluorescence staining, transmission electron microscopy, cell counting kit-8 assay, flow cytometry analysis, senescence-associated β-galactosidase staining, and western blotting, were accomplished to confirm the snRNA-seq results. We identified nine main cell types in adult ovaries and the cell-type-specific differentially expressed genes (DEGs) induced by CS exposure. Western blot results verified that down-regulation of antioxidant genes (Gpx1 and Wnt10b) and the steroid biosynthesis gene (Fdx1) occurred in both ovarian tissue and human granulosa cell-like tumor cell line (KGN cells) after CS exposure. Five percent cigarette smoke extract (CSE) effectively stimulated the production of reactive oxygen species (ROS), DNA damage, cellular senescence and markedly inhibited KGN cell proliferation by inducing G1-phase cell cycle arrest. Moreover, down-regulation of Gja1, Lama1 and the Ferroptosis indicator (Gpx4) in granulosa cells plays a significant role in ultrastructural changes in the ovary induced by CS exposure. These observations suggest that CS exposure impaired ovarian follicle reserve might be caused by REDOX imbalance in granulosa cells. The current study systematically determined the damage caused by CS in mouse ovaries and provides a theoretical basis for early clinical prediction, diagnosis and intervention of CS exposure-associated primary ovarian insufficiency (POI), and is of great significance in improving female reproductive health.

EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence.

Objectives Abdominal aortic aneurysm (AAA) has a high risk of rupture of the aorta and is one of the leading causes of death in older adults. This study is aimed at confirming the influence and mechanism of the abnormally expressed ANXA6 gene in AAA. Methods Clinical samples were collected for proteome sequencing to screen for differentially expressed proteins. An Ang II-induced vascular smooth muscle cell (VSMC) aging model as well as an AAA animal model was used. Using RT-qPCR to detect the mRNA levels of EZH2, ANXA6, IK-6, and IL-8 in cells and tissues were assessed. Western blotting and immunohistochemistry staining were used apply for the expression of associated proteins in cells and tissues. SA-β-gal staining, flow cytometry, and DHE staining were used to detect senescent cells and the level of ROS. The cell cycle was assessed by flow cytometry. Arterial pathology was observed by HE staining. The aging of VSMCs in arterial tissue was assessed by coimmunofluorescence for α-SMA and p53. Results There were 24 differentially expressed proteins in the AAA clinical samples, including 10 upregulated protein and 14 downregulated protein, and the differential expression of ANXA6 was associated with vascular disease. Our study found that ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced VSMC aging model. Knockdown of ANXA6 or overexpression of EZH2 inhibited Ang II-induced ROS, inhibited cell senescence, decreased Ang II evoked G1 arrest, and increased cells in G2 phase, while overexpression of ANXA6 played the opposite role. Overexpression of EZH2 inhibited ANXA6 expression by increasing H3K27me3 modification at the ANXA6 promoter. Simultaneous overexpression of EZH2 and the protective effect of EZH2 on cell senescence were partially reversed by ANXA6. Similarly, ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced AAA animal model. Knockdown of ANXA6 and overexpression of EZH2 alleviated Ang II-induced VSMC senescence and inhibited AAA progression, while simultaneous overexpression of EZH2 and ANXA6 partially reversed the protective effect of EZH2 on AAA. Conclusion EZH2 regulates the ANXA6 promoter H3K27me3 modification, inhibits ANXA6 expression, alleviates Ang II-induced VSMC senescence, and inhibits AAA progression.

Arbidol inhibits human esophageal squamous cell carcinoma growth in vitro and in vivo through suppressing ataxia telangiectasia and Rad3-related protein kinase.

Human esophageal cancer has a global impact on human health due to its high incidence and mortality. Therefore, there is an urgent need to develop new drugs to treat or prevent the prominent pathological subtype of esophageal cancer, esophageal squamous cell carcinoma (ESCC). Based upon the screening of drugs approved by the Food and Drug Administration, we discovered that Arbidol could effectively inhibit the proliferation of human ESCC in vitro. Next, we conducted a series of cell-based assays and found that Arbidol treatment inhibited the proliferation and colony formation ability of ESCC cells and promoted G1-phase cell cycle arrest. Phosphoproteomics experiments, in vitro kinase assays and pull-down assays were subsequently performed in order to identify the underlying growth inhibitory mechanism. We verified that Arbidol is a potential ataxia telangiectasia and Rad3-related (ATR) inhibitor via binding to ATR kinase to reduce the phosphorylation and activation of minichromosome maintenance protein 2 at Ser108. Finally, we demonstrated Arbidol had the inhibitory effect of ESCC in vivo by a patient-derived xenograft model. All together, Arbidol inhibits the proliferation of ESCC in vitro and in vivo through the DNA replication pathway and is associated with the cell cycle.

O-GlcNAc transferase is important for homology-directed repair

O-Linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.

Differentiation Capacity of Bone Marrow-Derived Rat Mesenchymal Stem Cells from DsRed and Cre Transgenic Cre/loxP Models

Cre/loxP recombination is a well-established technique increasingly used for modifying DNA both in vitro and in vivo. Nucleotide alterations can be edited in the genomes of mammalian cells, and genetic switches can be designed to target the expression or excision of a gene in any tissue at any time in animal models. In this study, we propose a system which worked via the Cre/loxP switch gene and DsRed/emGFP dual-color fluorescence imaging. Mesenchymal stem cells (MSCs) can be used to regenerate damaged tissue because of their differentiation capacity. Although previous studies have presented evidence of fusion of transplanted MSCs with recipient cells, the possibility of fusion in such cases remains debated. Moreover, the effects and biological implications of the fusion of MSCs at the tissue and organ level have not yet been elucidated. Thus, the method for determining this issue is significant and the models we proposed can illustrate the question. However, the transgenic rats exhibited growth slower than that of wild-type rats over several weeks. The effects on the stemness, proliferation, cell cycle, and differentiation ability of bone marrow–derived rat MSCs (BM-rMSCs) from the models were examined to ensure our design was appropriate for the in vivo application. We demonstrated that MSC surface markers were maintained in DsRed and Cre transgenic rMSCs (DsRed-rMSCs and Cre-rMSCs, respectively). A WST-8 assay revealed decreased proliferative activity in these DsRed-rMSCs and Cre-rMSCs; this result was validated through cell counting. Furthermore, cell cycle analysis indicated a decrease in the proportion of G1-phase cells and a concomitant increase in the proportion of S-phase cells. The levels of cell cycle–related proteins also decreased in the DsRed-rMSCs and Cre-rMSCs, implying decelerated phase transition. However, the BM-rMSCs collected from the transgenic rats did not exhibit altered adipogenesis, osteogenesis, or chondrogenesis. The specific markers of these types of differentiation were upregulated after induction. Therefore, BM-rMSCs from DsRed and Cre transgenic models can be used to investigate the behavior of MSCs and related mechanisms. Such application may further the development of stem cell therapy for tissue damage and other diseases.

TGF-β generates a population of cancer cells residing in G1 phase with high motility and metastatic potential via KRTAP2-3.

Transforming growth factor β (TGF-β) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-β also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-β confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-β, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-β generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-β-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis.

GASP1 enhances malignant phenotypes of breast cancer cells and decreases their response to paclitaxel by forming a vicious cycle with IGF1/IGF1R signaling pathway

There is a potential correlation between G-protein-coupled receptor-associated sorting protein 1 (GASP1) and breast tumorigenesis. However, its biological function and underlying molecular mechanism in breast cancer have not been clearly delineated. Here, we demonstrated that GASP1 was highly expressed in breast cancers, and patients harboring altered GASP1 showed a worse prognosis than those with wild-type GASP1. Functional studies showed that GASP1 knockout significantly suppressed malignant properties of breast cancer cells, such as inhibition of cell proliferation, colony formation, migration, invasion and xenograft tumor growth in nude mice as well as induction of G1-phase cell cycle arrest, and vice versa. Mechanistically, GASP1 inhibited proteasomal degradation of insulin-like growth factor 1 receptor (IGF1R) by competitively binding to IGF1R with ubiquitin E3 ligase MDM2, thereby activating its downstream signaling pathways such as NF-κB, PI3K/AKT, and MAPK/ERK pathways given their critical roles in breast tumorigenesis and progression. IGF1, in turn, stimulated GASP1 expression by activating the PI3K/AKT pathway, forming a vicious cycle propelling the malignant progression of breast cancer. Besides, we found that GASP1 knockout obviously improved the response of breast cancer cells to paclitaxel. Collectively, this study demonstrates that GASP1 enhances malignant behaviors of breast cancer cells and decreases their cellular response to paclitaxel by interacting with and stabilizing IGF1R, and suggests that it may serve as a valuable prognostic factor and potential therapeutic target in breast cancer.

Cytotoxic Effect of the Essential oils from Erigeron Canadensis L. on Human Cervical Cancer HeLa Cells in Vitro.

Erigeron Canadensis L. (E. canadensis) is a widely distributed invasive weed species in China. Potentially anti-cancer qualities may exist in its essential oils (EOs). The purpose of this study was to analyze the components of the EOs of E. canadensis and their effects on the normal liver cell lines L02 and the human cervical cancer cell lines HeLa. The EOs from the upper region of E. canadensis were prepared, its components were identified by GC/MS. Cell viability, cell morphology observation, AO/EB dual fluorescence staining assay, flow cytometry, mitochondrial membrane potential, western blot, caspase inhibitor test, and oxidative stress tests were used to investigate the impact of the EOs on HeLa cells. Network pharmacological analysis was employed to study the potential mechanism of the EOs in the treatment of cervical cancer. According to the findings, the EOs had 21 chemical components, of which limonene made up 65.68 %. After being exposed to the EOs, the cell viability of HeLa and L02 dramatically declined. The inhibition of EOs was more effective than that of limonene when used in an amount equivalent to that in the EOs. L02 cells were less susceptible to the cytotoxicity of EOs than HeLa cells were. Furthermore, EOs altered the cell cycle in HeLa cells and caused oxidative stress and apoptosis. Compared with the control group, the reactive oxygen species (ROS) levels increased in HeLa cells at first and then decreased, total superoxide dismutase (SOD) and catalase (CAT) activities in HeLa cells significantly decreased. G1 phase cells decreased whereas G2/M phase cells increased. The rate of apoptosis rose. Reduced mitochondrial membrane potential and Caspase-3, -9, and -12 protein expression were both observed. Nerolidol, dextroparaffinone, and α-pinene were shown to be the primary components for the suppression of HeLa cells, according to the results of the prediction of pharmacologic targets. In conclusion, findings of this study indicated the EOs may have the potential to curb the growth of cervical cancer cells. Further research is needed to explore the in vivo effect of EOs.

Loss of VOPP1 Contributes to BET Inhibitor Acquired Resistance in Non-Small Cell Lung Cancer Cells.

Based on these results, for the first time, we establish a causal link from VOPP1 loss to BCL-2 gain and then to BETi resistance, which provides new insights into BETi resistance and paves the way for further testing to circumvent BETi resistance.

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells.

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells.

Growth conditions inducing G1 cell cycle arrest enhance lipid production in the oleaginous yeast Lipomyces starkeyi.

Lipid droplets are cytoplasmic organelles that store lipids for energy and membrane synthesis. The oleaginous yeast Lipomyces starkeyi is one of the most promising lipid producers and has attracted attention as a biofuel source. It is known that the expansion of lipid droplets is enhanced under nutrient-poor conditions. Therefore, we prepared a novel nitrogen-depleted medium (N medium) in which to culture L. starkeyi cells. Lipid accumulation was rapidly induced, and this was reversed by the addition of ammonium. In this condition, cell proliferation stopped, and cells with giant lipid droplets were arrested in G1 phase. We investigated whether cell cycle arrest at a specific phase is required for lipid accumulation. Lipid accumulation was repressed in hydroxyurea-synchronized S phase cells and was increased in nocodazole-arrested G2/M phase cells. Moreover, the enrichment of G1 phase cells seen upon rapamycin treatment induced massive lipid accumulation. From these results, we conclude that L. starkeyi cells store lipids from G2/M phase and then arrest cell proliferation in the subsequent G1 phase, where lipid accumulation is enhanced. Cell cycle control is an attractive approach for biofuel production.

Duplication and Segregation of Centrosomes during Cell Division.

During its division the cell must ensure the equal distribution of its genetic material in the two newly created cells, but it must also distribute organelles such as the Golgi apparatus, the mitochondria and the centrosome. DNA, the carrier of heredity, located in the nucleus of the cell, has made it possible to define the main principles that regulate the progression of the cell cycle. The cell cycle, which includes interphase and mitosis, is essentially a nuclear cycle, or a DNA cycle, since the interphase stages names (G1, S, G2) phases are based on processes that occur exclusively with DNA. However, centrosome duplication and segregation are two equally important events for the two new cells that must inherit a single centrosome. The centrosome, long considered the center of the cell, is made up of two small cylinders, the centrioles, made up of microtubules modified to acquire a very high stability. It is the main nucleation center of microtubules in the cell. Apart from a few exceptions, each cell in G1 phase has only one centrosome, consisting in of two centrioles and pericentriolar materials (PCM), which must be duplicated before the cell divides so that the two new cells formed inherit a single centrosome. The centriole is also the origin of the primary cilia, motile cilia and flagella of some cells.

Daurisoline Inhibits ESCC by Inducing G1 Cell Cycle Arrest and Activating ER Stress to Trigger Noxa-Dependent Intrinsic and CHOP-DR5-Dependent Extrinsic Apoptosis via p-eIF2α-ATF4 Axis.

Esophageal squamous cell carcinoma (ESCC), one of the most malignant human cancers in clinic, requires novel treatment. Daurisoline (DAS) is a component of traditional Chinese herb, which exhibits anti-cancer effects by autophagy inhibition and metastasis suppression. However, the effect and mechanism of DAS on ESCC remain unclear. Here, we found that DAS inhibited cell proliferation and colony formation in both human ESCC cell lines EC1 and ECA109. Mechanistically, DAS induced p21-/p27-dependent G1 phase cell cycle arrest and apoptosis in a dose-dependent manner. The induction of apoptosis by DAS was largely dependent on the activation of the transcription factor ATF4 and its downstream NOXA-dependent intrinsic and CHOP-DR5-dependent extrinsic apoptotic pathway. ATF4 activation induced by DAS was due to the generation of excessive reactive oxygen species (ROS) and the subsequent activation of endoplasmic reticulum (ER) stress through the p-eIF2α-ATF4 signal pathway, which can be largely abrogated by N-acetylcysteine (NAC), a scavenger of ROS. Moreover, DAS treatment significantly inhibited tumor growth and reduced tumor weight in the tumor xenograft mouse model by up-regulating key proteins related to cell cycle arrest and apoptotic pathway. Taken together, these findings identified DAS as a novel candidate for the treatment of ESCC.

Bone morphogenetic protein 4 rescues the bone regenerative potential of old muscle-derived stem cells via regulation of cell cycle inhibitors

BackgroundBone morphogenetic protein 4 (BMP4) promotes the osteogenic differentiation and the bone regenerative potential of muscle-derived stem cells (MDSCs). BMP4 also promotes the self-renewal of both embryonic and somatic stem cells; however, BMP4 signaling activity significantly decreases with age. Cyclin-dependent kinase inhibitors P16INK4A (P16) and P18INK4C (P18) induce early G1-phase cell cycle blockade by targeting cyclin-dependent kinase 4/6. It is still unclear if BMP4 affects the bone regenerative potential of old MDSCs through regulation of P16 and P18 expression.MethodsYoung and old MDSCs were isolated from 3 week (young) and 2-year-old (old) mice. In vitro cell proliferation and multipotent differentiation were performed for young and old MDSCs both before and after BMP4/GFP transduction. Cell cycle genes were analyzed using Q-PCR. The bone regenerative potential of young and old MDSCs transduced with BMP4/GFP were compared using Micro-CT and histological analysis. The bone regenerative potential of young and old MDSCs was also compared between single and double transduction (higher BMP4 levels expression). The cell proliferation, mitochondrial function and osteogenic differentiation was also compared in vitro between cells that have been transduced with BMP4GFP (single and double transduction). The correlation of bone regeneration capacity of young and old MDSCs with P16 and P18 expression was further evaluated at 10 days after cell transplantation using histology and western blot analysis.ResultsOld murine MDSCs (MDSCs) exhibit reduced proliferation and multi-lineage differentiation potential with or without BMP4 stimulation, when compared to young murine MDSCs. Old MDSCs express significantly higher P16 and lower P18, with more cells in the G0/1 phase and fewer cells in the G2/M phase, compared to young MDSCs. Old MDSCs retrovirally transduced to express BMP4 regenerated less bone in a critical size skull defect in CD-1 nude mice when compared to young retrovirally transduced MDSCs expressing similar BMP4 levels and contribute less to the new regenerated new bone. Importantly, both young and old MDSCs can regenerate more bone when BMP4 expression levels are increased by double-transduction with the retroviral-BMP4/GFP. However, the bone regeneration enhancement with elevated BMP4 was more profound in old MDSCs (400% at 2 weeks) compared to young MDSCs (200%). Accordingly, P18 is upregulated while P16 is downregulated after BMP4 transduction. Double transduction did not further increase cell proliferation nor mitochondrial function but did significantly increase Osx expression in both young and old MDSCs. Old MDSCs had even significant higher Osx levels as compared to young MDSCs following double transduction, while a similar Alp expression was observed between young and old MDSCs after double transduction. In addition, at 10 days after cell transplantation, old MDSCs having undergone double transduction regenerated bone more rapidly as showed by Alcian blue and Von Kossa staining. Western blot assays demonstrated that old MDSCs after retro-BMP4/GFP double transduction have significantly lower P18 expression levels when compared to young BMP4-transduced MDSCs. In addition, P18 expression was slightly increased in old MDSCs after double transduction when compared to single transduction. P16 expression was not detectable for both young and two old BMP4/GFP transduced MDSCs groups.ConclusionsIn summary, BMP4 can offset the adverse effect of aging on the osteogenic differentiation and the bone regenerative potential of old MDSCs via up-regulation of P18 and down-regulation P16 expression.