Caffeine causes cell cycle arrest at G0/G1 and increases of ubiquitinated proteins, ATP and mitochondrial membrane potential in renal cells
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PROVIDER: S-EPMC10550404 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:Caffeine is a well-known purine alkaloid commonly found in coffee. Several lines of previous and recent evidence have shown that habitual coffee drinking is associated with lower risks for chronic kidney disease (CKD) and nephrolithiasis. However, cellular and molecular mechanisms underlying its renoprotective effects remain largely unknown due to a lack of knowledge on cellular adaptive response to caffeine. This study investigated cellular adaptive response of renal tubular cells to caffeine at the protein level. Cellular proteome of MDCK cells treated with caffeine at a physiologic concentration (100 μM) for 24 h was analyzed comparing with that of untreated cells by label-free quantitative proteomics. From a total of 936 proteins identified, comparative analysis revealed significant changes in levels of 148 proteins induced by caffeine. These significantly altered proteins were involved mainly in proteasome, ribosome, tricarboxylic acid (TCA) (or Krebs) cycle, DNA replication, spliceosome, biosynthesis of amino acid, carbon metabolism, nucleocytoplasmic transport, cell cycle, cytoplasmic translation, translation initiation, and mRNA metabolic process. Functional validation by various assays confirmed that caffeine decreased cell population at G2/M, increased cell population at G0/G1, increased level of ubiquitinated proteins, increased intracellular ATP and enhanced mitochondrial membrane potential in MDCK cells. These data may help unravelling molecular mechanisms underlying the biological effects of caffeine on renal tubular cells at cellular and protein levels.
2023-12-11 | PXD045313 | Pride
Project description:Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus, has caused enormous economic losses in the swine industry. p53 protein exists in a wide variety of animal cells, which is involved in cell cycle regulation, apoptosis, cell differentiation and other biological functions. In this study, we investigated the effects of PEDV infection on the cell cycle of Vero cells and p53 activation. The results demonstrated that PEDV infection induces cell cycle arrest at G0/G1 phase in Vero cells, while UV-inactivated PEDV does not cause cell cycle arrest. PEDV infection up-regulates the levels of p21, cdc2, cdk2, cdk4, Cyclin A protein and down-regulates Cyclin E protein. Further research results showed that inhibition of p53 signaling pathway can reverse the cell cycle arrest in G0/G1 phase induced by PEDV infection and cancel out the up-regulation of p21 and corresponding Cyclin/cdk mentioned above. In addition, PEDV infection of the cells synchronized in various stages of cell cycle showed that viral subgenomic RNA and virus titer were higher in the cells released from G0/G1 phase synchronized cells than that in the cells released from the G1/S phase and G2/M phase synchronized or asynchronous cells after 18?h p.i.. This is the first report to demonstrate that the p53-dependent pathway plays an important role in PEDV induced cell cycle arrest and beneficially contributes to viral infection.
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Project description:UnlabelledMany viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae.ImportanceNoroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1 phase for RNA virus replication.
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Project description:Acanthopanax senticosus (Rupr. et Maxim) Harms (ASH), also known as Siberian ginseng or eleuthero, is a hardy shrub native to China, Korea, Russia and the northern region of Japan. ASH is used for the treatment of several diseases such as heart disease, hypertension, rheumatoid arthritis, allergies, chronic bronchitis, diabetes and cancer. In the present study, the inhibitory effect of the root extract of ASH (ASHE) on HuH‑7 and HepG2 liver cancer cells was examined. ASHE suppressed liver cancer cell proliferation by inducing cell cycle arrest at the G0/G1 phase, as well as apoptosis, as indicated by the increased number of Annexin V and 7‑AAD‑positive cells. Furthermore, the expression of LC3‑II, an autophagy marker, in these cells also increased post treatment with ASHE. LC3‑II induction was further enhanced by co‑treatment with chloroquine. Fluorescence and transmission electron micrographs of ASHE‑treated liver cancer cells showed the presence of an increased number of autophagic vesicles. A decreased protein expression level of run domain Beclin‑1‑interacting and cysteine‑rich domain‑containing, an autophagy inhibitor, with no change in RUBCN mRNA expression was observed, indicating activation of the autophagosome‑lysosome fusion step of autophagy. In conclusion, ASHE exerts cytostatic activity on liver cancer cells via both apoptosis and autophagy, and may serve as a potential therapeutic agent for management of liver cancer and autophagy‑related diseases.
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Project description:Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N-terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co-immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.
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Project description:ObjectivesKeloids are benign fibroproliferative tumors that display many cancer-like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo-like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids.Materials and methodsWe evaluated the expression of PLK4 in keloids and adjacent normal skin tissue samples. Then, we established PLK4 knockdown and overexpression cell lines in keloid fibroblasts (KFs) and normal skin fibroblasts (NFs), respectively, to investigate the roles of PLK4 in the regulation of proliferation, migration, invasion, apoptosis, and cell cycle in KFs. Centrinone B (Cen-B), a highly selective PLK4 inhibitor, was used to inhibit PLK4 activity in KFs to evaluate the therapeutic effect on KFs.ResultsWe discovered that PLK4 was overexpressed in keloid dermal samples and KFs compared with adjacent normal skin samples and NFs derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by Cen-B suppressed KF growth, induced KF apoptosis via the caspase-9/3 pathway, and induced cell cycle arrest at the G0/G1 phase in vitro.ConclusionsThese findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen-B is a promising candidate drug for keloid treatment.
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Project description:BackgroundSOX4 is a transcription factor required for tissue development and differentiation in vertebrates. Overexpression of SOX4 has been reported in many cancers including glioblastoma multiforme (GBM), however, the underlying mechanism of actions has not been studied. In this study, we investigated the role of SOX4 in GBM.MethodsKaplan-Meier analysis was performed to assess the association between SOX4 expression levels and survival times in primary GBM samples. Cre/lox P system was used to generate gain or loss of SOX4 in GBM cells, and microarray analysis uncovered the regulation network of SOX4 in GBM cells.ResultsHigh SOX4 expression was significantly associated with good prognosis of primary GBMs. SOX4 inhibited the growth of GBM cell line LN229, A172G and U87MG, partly via the activation of p53-p21 signaling and down-regulation of phosphorylated AKT1. Gene expression profiling and subsequent gene ontology analysis showed that SOX4 influenced several key pathways including the Wnt/ beta-catenin and TGF-beta signaling pathways.ConclusionsOur study found that SOX4 acts as a tumor suppressor in GBM cells by induce cell cycle arrest and inhibiting cell growth.
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Project description:CDK4 is highly expressed and associated with poor prognosis and decreased survival in advanced neuroblastoma (NB). Targeting CDK4 degradation presents a potentially promising therapeutic strategy compared to conventional CDK4 inhibitors. However, the autophagic degradation of the CDK4 protein and its anti-proliferation effect in NB cells has not been mentioned. We identified autophagy as a new pathway for the degradation of CDK4. Firstly, autophagic degradation of CDK4 is critical for NVP-BEZ235-induced G0/G1 arrest, as demonstrated by the overexpression of CDK4, autophagy inhibition, and blockade of autophagy-related genes. Secondly, we present the first evidence that p62 binds to CDK4 and then enters the autophagy-lysosome to degrade CDK4 in a CTSB-dependent manner in NVP-BEZ235 treated NB cells. Similar results regarding the interaction between p62 and CDK4 were observed in the NVP-BEZ235 treated NB xenograft mouse model. Autophagic degradation of CDK4 plays a pivotal role in G0/G1 cell cycle arrest in NB cells treated with NVP-BEZ235.
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Project description:The core components of regenerative medicine are stem cells with high self-renewal and tissue regeneration potentials. Adult stem cells can be obtained from many organs and tissues. NANOG, SOX2 and OCT4 represent the core regulatory network that suppresses differentiation-associated genes, maintaining the pluripotency of mesenchymal stem cells. The roles of NANOG in maintaining self-renewal and undifferentiated status of adult stem cells are still not perfectly established. In this study we define the effects of downregulation of NANOG in maintaining self-renewal and undifferentiated state in mesenchymal stem cells (MSCs) derived from subcutaneous adipose tissue (hASCs). hASCs were expanded and transfected in vitro with short hairpin Lentivirus targeting NANOG. Gene suppressions were achieved at both transcript and proteome levels. The effect of NANOG knockdown on proliferation after 10 passages and on the cell cycle was evaluated by proliferation assay, colony forming unit (CFU), qRT-PCR and cell cycle analysis by flow-cytometry. Moreover, NANOG involvement in differentiation ability was evaluated. We report that downregulation of NANOG revealed a decrease in the proliferation and differentiation rate, inducing cell cycle arrest by increasing p27/CDKN1B (Cyclin-dependent kinase inhibitor 1B) and p21/CDKN1A (Cyclin-dependent kinase inhibitor 1A) through p53 and regulate DLK1/PREF1. Furthermore, NANOG induced downregulation of DNMT1, a major DNA methyltransferase responsible for maintaining methylation status during DNA replication probably involved in cell cycle regulation. Our study confirms that NANOG regulates the complex transcription network of plasticity of the cells, inducing cell cycle arrest and reducing differentiation potential.
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Project description:While having already killed more than 7 million of people worldwide in 4 years, SARS-CoV-2, the etiological agent of COVID-19, is still circulating and evolving. Understanding the pathogenesis of the virus is of capital importance. It was shown that in vitro and in vivo infection with SARS-CoV-2 can lead to cell cycle arrest but the effect of the cell cycle arrest on the virus infection and the associated mechanisms are still unclear. By stopping cells in the G1 phase as well as targeting several pathways involved using inhibitors and small interfering RNAs, we were able to determine that the cell cycle arrest in the late G1 is beneficial for SARS-CoV-2 replication. This cell cycle arrest is independent of p53 but is dependent on the CDC25A-CDK2/cyclin E pathway. These data give a new understanding in SARS-CoV-2 pathogenesis and highlight some possible targets for the development of novel therapeutic approaches.
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