An in vitro attempt at precision toxicology reveals the involvement of DNA methylation alteration in ochratoxin A-induced G0/G1 phase arrest.
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ABSTRACT:
SUBMITTER: Zhang B
PROVIDER: S-EPMC6961687 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Electron transport chain (ETC) is responsible for oxidative phosphorylation-mediated mitochondrial respiration. Here we wanted to address the mahanine-induced targeted pathways in glioblastoma multiforme (GBM) in the context of G0/G1 phase arrest and redox alteration. We have demonstrated mahanine, as a novel mitochondrial complex-III inhibitor which induced G0/G1 phase arrest in GBM. This event was preceded by accumulation of intracellular ROS by the inhibition of mitochondrial ETC. The accumulated ROS induced DNA damage response (DDR), that mediated Chk1/Chk2 upregulation and activation which were essential factors for the G0/G1 arrest. NAC-mediated scavenging of ROS generation reduced the propensity of G0/G1 phase arrest in GBM cells by mahanine. Knockdown of Chk1/Chk2 also affected the cell cycle inhibitory potential of mahanine. During G0/G1 arrest, other hallmark proteins like, cyclin D1/cyclin D3, CDK4/CDK6 and CDC25A were also downregulated. The G0/G1 phase restriction property of mahanine was also established in in vivo mice model. Mahanine-induced complex-III inhibition triggered enhanced ROS in hypoxia responsible for higher G0/G1 arrest. Furthermore, we demonstrated that mahanine-treated G0/G1 arrested cells were less potent to form xenograft tumor in vivo. Additionally, they exhibited reduced ability to migrate and form intracellular tube-like structures. Moreover, they became susceptible to differentiate and astrocyte-like cells were generated from the epithelial lineage. Taken together, our results established that complex-III of ETC is one of the possible potential targets of mahanine. This nontoxic chemotherapeutic molecule enhanced ROS production, induced cell cycle arrest and thereafter regressed GBM without effecting normal astrocytes.
| S-EPMC4266700 | biostudies-literature
Project description:Many viruses exploit the host cell division cycle to favour their own growth. Here we demonstrated that porcine circovirus type 2 (PCV2), which is a major causative agent of an emerging and important swine disease complex, PCV2-associated diseases, caused G0/G1 cell cycle arrest through degradation of cyclin D1 and E followed by reduction of retinoblastoma phosphorylation in synchronized PCV2-infected cells dependent upon virus replication. This induction of G0/G1 cell cycle arrest promoted PCV2 replication as evidenced by increased viral protein expression and progeny virus production in the synchronized PCV2-infected cells. To delineate a mechanism of miRNAs in regulating PCV2-induced G0/G1 cell cycle arrest, we determined expression levels of some relevant miRNAs and found that only miR-15a but not miR-16, miR-21, and miR-34a was significantly changed in the PCV2-infected cells. We further demonstrated that upregulation of miR-15a promoted PCV2-induced G0/G1 cell cycle arrest via mediating cyclins D1 and E degradation, in which involves PCV2 growth. These results reveal that G0/G1 cell cycle arrest induced by PCV2 may provide favourable conditions for viral protein expression and progeny production and that miR-15a is implicated in PCV2-induced cell cycle control, thereby contributing to efficient viral replication.
| S-EPMC4908419 | biostudies-other
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.
| S-EPMC4442456 | biostudies-literature
Project description:PurposeCytoskeleton is critical for carcinoma cell proliferation, migration, and invasion. Sad-1 and UNC-84 domain containing 1 (SUN1) is one of the core linkers of nucleoskeleton and cytoskeleton. However, the functions of SUN1 in lung adenocarcinoma are largely unknown.MethodsIn this study, we first transduced the lentivirus delivering the short hairpin RNA (shRNA) against SUN1 to lung adenocarcinoma cells (A549 and 95D cells) with high efficiency. After lentivirus infection, quantitative real-time polymerase chain reaction and Western blotting were used to detect the expressions of SUN1 mRNA and protein. The cell proliferation and colony formation were detected by MTT assay and colony formation assay, respectively. The cell distribution in the cell cycle was analyzed by flow cytometry.ResultsBoth mRNA and protein levels of SUN1 were significantly decreased in A549 and 95D cells after lentivirus infection, as indicated by quantitative real-time polymerase chain reaction and Western blot. Next, we found that cell proliferation and colony formation were markedly reduced in SUN1 silenced cells. Moreover, suppression of SUN1 led to cell cycle arrest at G0/G1 phase. Furthermore, Cyclin D1, CDK6, and CDK2 expressions were obviously reduced in A549 cells after SUN1 silencing.ConclusionThese results suggest that SUN1 plays an essential role in proliferation of lung adenocarcinoma cells in vitro and may be used as a potential therapeutic target for the treatment of lung adenocarcinoma in the future.
| S-EPMC5476752 | biostudies-literature
Project description:BackgroundDepletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear.MethodsExpression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo.ResultsWe discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model.ConclusionOur findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.
| S-EPMC3639102 | biostudies-literature
Project description:BackgroundTo assess the level of CR6-interacting factor 1 (CRIF1), a cell cycle negative regulator, in patients with leukemia and investigate the role of CRIF1 in regulating leukemia cell cycle.MethodsWe compared the CRIF1 level in bone marrow (BM) samples from healthy and acute myeloid leukemia (AML), iron deficiency anemia (IDA) and AML-complete remission (AML-CR) subjects. We also manipulated CRIF1 level in the Jurkat cells using lentivirus-mediated overexpression or siRNA-mediated depletion. Co-culture with the BM stromal cells (BMSCs) was used to induce leukemia cell cycle arrest and mimic the BM microenvironment.ResultsWe found significant decreases of CRIF1 mRNA and protein in the AML group. CRIF1 overexpression increased the proportion of Jurkat cells arrested in G0/G1, while depletion of endogenous CRIF1 decreased cell cycle arrest. Depletion of CRIF1 reversed BMSCs induced cell cycle arrest in leukemia cells. Co-immunoprecipitation showed a specific binding of CDK2 to CRIF1 in Jurkat cells during cell cycle arrest. Co-localization of two proteins in both nucleus and cytoplasm was also observed with immunofluorescent staining.ConclusionCRIF1 may play a regulatory role in the BM microenvironment-induced leukemia cell cycle arrest possibly through interacting with CDK2 and acting as a cyclin-dependent kinase inhibitor.
| S-EPMC3919709 | biostudies-literature
Project description:Both preclinical and epidemiology studies associate ?-adrenoceptors-blockers (?-blockers) with activity against melanoma. However, the underlying mechanism is still unclear, especially in acral melanoma. In this study, we explored the effect of propranolol, a non-selective ?-blocker, on the A375 melanoma cell line, two primary acral melanoma cell lines (P-3, P-6) and mice xenografts. Cell viability assay demonstrated that 50?M-400?M of propranolol inhibited viability in a concentration and time dependent manner with an IC50 ranging from 65.33?M to 148.60?M for 24h -72h treatment, but propranolol (less than 200?M) had no effect on HaCaT cell line. Western blots showed 100?M propranolol significantly reduced the expression of Bcl-2 while increasing the expressions of Bax, cytochrome c, cleaved capase-9 and cleaved caspase-3, and down-regulated the levels of p-AKT, p-BRAF, p-MEK1/2 and p-ERK1/2 in melanoma cells, after a 24h incubation. The in vivo data confirmed the isolation results. Mice received daily ip. administration of propranolol at the dose of 2 mg/kg for 3 weeks and the control group was treated with the same volume of saline. The mean tumor volume at day 21 in A375 xenografts was 82.33 ± 3.75mm3vs. 2044.67 ± 54.57mm3 for the propranolol-treated mice and the control group, respectively, and 31.66 ± 4.67 mm3vs. 1074.67 ± 32.17 mm3 for the P-3 xenografts. Propranolol also reduced Ki67, inhibited phosphorylation of AKT, BRAF, MEK1/2 and ERK1/2 in xenografts. These are the first data to demonstrate that propranolol might inhibit melanoma by activating the intrinsic apoptosis pathway and inactivating the MAPK and AKT pathways.
| S-EPMC5356557 | biostudies-literature
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.
| S-EPMC9251049 | biostudies-literature
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.
| S-EPMC4233052 | biostudies-literature
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.
| S-EPMC6593979 | biostudies-literature