A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

2016 ◽  
Vol 42 (4) ◽  
pp. 997-1005 ◽  
Author(s):  
Shi-Jun Zhao ◽  
Xian-Jun Wang ◽  
Qing-Jian Wu ◽  
Chao Liu ◽  
Da-Wei Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Glioma Cells ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

scholarly journals A RAD9-dependent cell cycle arrest in response to unresolved recombination intermediates in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Hardeep Kaur ◽  
GN Krishnaprasad ◽  
Michael Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Cell Cycle Arrest ◽  
Mitotic Cell ◽  
Cycle Arrest ◽  
Associated Lesions

AbstractIn Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are precursors of crossovers. In vitro studies have suggested that the dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation could be responsible for this. To ask if dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth, a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during return to growth delayed joint molecule resolution, but ultimately most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9Δ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in rad9Δ, Rmi1-depleted cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

scholarly journals In Vitro and In Vivo Activity of Fomitopsis Pinicola (Sw. Ex Fr.) Karst Chloroform (Fpkc) Extract Against S180 Tumor Cells

2017 ◽  
Vol 44 (5) ◽  
pp. 2042-2056 ◽  
Author(s):  
Ye Gao ◽  
Pan Wang ◽  
Yaqin Wang ◽  
Lijie Wu ◽  
Xiaobing Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Survival Time ◽  
Tumor Cells ◽  
Propidium Iodide ◽  
Cell Morphology ◽  
Fomitopsis Pinicola ◽  
Cycle Arrest

Background/Aims: Non-toxic fomitopsis is has been traditionally used in folk medicine in many countries for its anti-inflammatory and anti-vascular disease activities. The present study investigates the antitumor effect of Fomitopsis pinicola (Sw. Ex Fr.) Karst chloroform extract (FPKc) on S180 tumor cells in vitro and in vivo and we determined the underlying mechanisms. Methods: HPLC was employed to analyze the constituents of FPKc. In-vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to quantify the growth inhibition of FPKc; Propidium iodide (PI) exclusion assay and scanning electron microscopy (SEM) were used to observe the damage on the cell membrane and the changes of the cell morphology; Staining with Hoechst 33342/propidium iodide (HO/PI) and the application of the Annexin V-FITC/PI analysis permitted to observe the cell death triggered by FPKc; DNA damage and cell cycle arrest were detected by flow cytometry; Rhodamine 123 (RH123) and Cytochrome C were used as dyes to investigate the alterations of the mitochondria. In-vivo tumor inhibition and mice survival time were determined. Results: The results of the HPLC assay indicated that FPKc might contain DA (dehydroeburiconic acid), PA (pachymic acid), and ES (ergosterol), at percentages of 0.25%, 17.8%, and 10.5%, respectively. Concerning the study of the biological function, the results showed that FPKc exhibited preferential and significant suppression of proliferation on specific cell lines including S180, HL-60, U937, K562, SMMC-7721, and Eca-109 cells, which induced plasma membrane and cell morphology damages, triggering S180 tumor-cells late apoptosis and leading to DNA damage and S phase arrest. Mitochondria were suspected to play a vital role in these changes. In vivo data indicated that FPKc inhibited the solid tumor growth and prolonged the survival time of tumor-bearing mice. Moreover, FPKc provoked only little damage on normal cells in vitro and also on normal tissues in vivo. Conclusion: FPKc inhibited S180 tumor cells growth and prolonged the lifespan of mice. In vitro, we found that FPKc induced S180 tumor cells apoptosis and cell cycle arrest, possibly via the mitochondrial pathway.

scholarly journals DIPG-53. CHARACTERIZING THE ROLE OF PPM1D MUTATIONS IN THE PATHOGENESIS OF DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGS)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii297-iii297
Author(s):  
Prasidda Khadka ◽  
Zachary Reitman ◽  
Sophie Lu ◽  
Graham Buchan ◽  
Rachel Hartley ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Radiation Treatment ◽  
Ectopic Expression ◽  
Loss Of Function ◽  
In Vivo Models ◽  
Cycle Arrest ◽  
Diffuse Intrinsic Pontine Gliomas

Abstract INTRODUCTION We have previously found that up to 15% of all DIPGs harbor mutations in PPM1D, resulting in the expression of an activated and truncated PPM1D (PPM1Dtr). Here we evaluate the mechanisms through which PPM1Dtr enhances glioma formation and identify its associated therapeutic vulnerabilities. METHODS We have developed multiple in vitro and in vivo models of PPM1D-mutant DIPGs and applied quantitative proteomic and functional genomic approaches to identify pathways altered by PPM1Dtr and associated dependencies. RESULTS PPM1D mutations are clonal events that are anti-correlated to TP53 mutations. We find ectopic expression of PPM1Dtr to be sufficient to enhance glioma formation and to be necessary in PPM1D-mutant DIPG cells. In addition, endogenous truncation of PPM1D is sufficient to enhance glioma formation in the presence of mutant H3F3A and PDGFRA. PPM1Dtr overexpression attenuates g-H2AX formation and suppresses apoptosis and cell-cycle arrest in response to radiation treatment. Deep scale phosphoproteomics analyses reveal DNA-damage and cell cycle pathways to be most significantly associated with PPM1Dtr. Furthermore, preliminary analysis of genome-wide loss-of-function CRISPR/Cas9 screens in isogenic GFP and PPM1Dtr overexpressing mouse neural stem cells reveal differential dependency on DNA-damage response genes in the PPM1Dtr overexpressing cells. Consistent with PPM1D’s role in stabilizing MDM2, PPM1D-mutant DIPG models are sensitive to a panel of MDM2 inhibitors (Nutlin-3a, RG7388, and AMG232). CONCLUSION Our study shows that PPM1Dtr is both an oncogene and a dependency in PPM1D- mutant DIPG, and there are novel therapeutic vulnerabilities associated with PPM1D that may be exploited.

scholarly journals Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53

2000 ◽  
Vol 14 (3) ◽  
pp. 278-288 ◽  
Author(s):  
Nabil H. Chehab ◽  
Asra Malikzay ◽  
Michael Appel ◽  
Thanos D. Halazonetis
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Cycle Arrest ◽  
P53 Tumor Suppressor Protein

Chk2/hcds1, the human homolog of theSaccharomyces cerevisiae RAD53/SPK1 andSchizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G2 in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G1 arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G1.

Inhibition Of Telomerase Is a Novel and Effective Therapy In MLL-Rearranged Acute Myeloid Leukemia (AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2887-2887
Author(s):  
Claudia Bruedigam ◽  
Frederik Otzen Bagger ◽  
Catherine Paine Kuhn ◽  
Therese Vu ◽  
Rebecca Austin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Prognostic Impact ◽  
Hematopoietic Stem ◽  
Telomerase Inhibition ◽  
Replicative Stress ◽  
Cycle Arrest

Abstract Telomerase is activated to maintain the long-term replicative potential in many human cancers including AML and novel inhibitors of telomerase have recently entered clinical trials for a variety of malignancies. We investigated the therapeutic potential of telomerase inhibition on MLL-rearranged AML using genetic and pharmacological approaches in murine and humanized models. Telomerase-deficient AML was generated by retroviral transduction of G3 Terc-/- LKS+ (Lin-Kit+Sca1+, enriched for hematopoietic stem cells) with pMIG-MLL-AF9 and compared to wild type (WT) controls. Transformed Terc-/- LKS+ colony-forming units (CFU) were mildly reduced at early passage (week 1 Terc-/- 13.1 ± 1 vs. WT 32.7 ± 4 per 1000 cells input, p < 0.01) but became progressively extinguished with serial replating (week 6 Terc-/- 3.8 ± 0.4 vs. WT 27.0 ± 2.7 per 1000 input, p < 0.01). Loss of CFU correlated with enforced differentiation (reduced Kit, increased Gr1), cell cycle arrest and preferential apoptosis of Kit+ cells. In vivo, AML developed with delayed latency, but was fully penetrant in recipients of G3 Terc-/- and WT cells (Terc-/- 64 days vs. WT 45 days, p < 0.01). Leukemic burden and leukemia stem cell (LSC, GFP+Lin-Sca1-Kit+FcgR+CD34-) frequency were similar between Terc-/- and WT AML. To determine the consequences of telomerase loss on AML LSCs, we performed gene expression profiling of purified LSCs. MLL-AF9-Terc-/- LSCs revealed enrichment of pathways controlling DNA damage/repair, cell cycle and apoptosis. Upstream analysis predicted activation of p53, Rbl1 and Cdkn2a, and inhibition of E2f1 in Terc-/- LSCs. Functionally, shRNA-mediated knockdown of p53 in Terc-/- LSCs partially rescued in vitro CFU, differentiation, cell cycle arrest and apoptosis. The phenotypic changes in Terc-/- AML were amplified by serial passage, suggesting that replicative stress may exacerbate the deleterious effects of telomerase loss on LSC function. To enforce replicative stress in vivo, we performed serial transplantation of Terc-/- AML vs. WT AML. Terc-/- LSCs were unable to generate secondary AML (survival Terc-/- not reached vs. WT 28 days, p < 0.01) and this was confirmed by limiting dilution analysis (Terc-/- LSC frequency 1:224,000 vs. WT 1:184 p < 0.001). Initial engraftment was similar between Terc-/- and WT LSCs. In vivo leukemogenesis was prevented by cell cycle arrest, DNA damage and massive apoptosis (Terc-/- 76.8% ± 3.6% vs. WT 22.49% ± 2.3%, p < 0.0001). Together, these findings demonstrate that in this murine model, telomerase loss eradicates LSC in vivo. To validate these findings in human AML we performed lentiviral shRNA knockdown of hTERT in the MLL-AF9-containing AML cell line Monomac6, followed by transplantation into NSGS (NOD/SCID/IL2Rgamma-/- transgenic for hSCF/hIL3/hGMCSF) xenograft recipients. Two independent shTERT constructs revealed significantly increased survival compared to non-transduced and non-targeting controls (sh-hTERT 149.5 days and 146 days vs. non-transduced 47 days, non-targeting 53.5 days, p< 0.01 for both sh-hTERT). hTERT knockdown correlated with reduced hCD45 engraftment, induction of DNA damage, cell cycle arrest and apoptosis compared to non-transduced or non-targeting shRNA controls. Pharmacological inhibition of telomerase (Telomerase Inhibitor IX, T-IX, Sigma) reduced growth of multiple human AML cell lines in vitro. Treatment with T-IX followed by transplantation of equal numbers of Monomac6 cells prolonged NSGS xenograft survival (T-IX 96.5 days vs. DMSO 59 days, p < 0.05). Finally, we examined the prognostic impact of telomerase-regulated genes in a large cohort of patients with AML. The top 140 differentially expressed genes (p < 0.001) between murine Terc-/- and WT LSCs predicted survival in 2 independent AML clinical trial cohorts. Computational modelling using a random forest approach was able to identify 10 key telomerase-regulated human homologues that could cluster AML patients into prognostically relevant groups and was reproducible in multiple independent datasets. These findings provide new mechanistic understanding into the effects of telomerase inhibition on MLL-rearranged AML and identify the telomerase complex as a novel therapeutic target for AML. Disclosures: No relevant conflicts of interest to declare.

The Hybrid Molecule, Edo-S101, Impairs Double Strand Breaks Repair in Multiple Myeloma and Synergizes with Bortezomib and Dexamethasone

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5354-5354 ◽  
Author(s):  
Ana Alicia López-Iglesias ◽  
Ana Belen Herrero ◽  
Laura San-Segundo ◽  
Susana Hernández-García ◽  
Lorena González-Méndez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Comet Assay ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Ex Vivo ◽  
Hybrid Molecule ◽  
Cycle Arrest

Abstract Introduction. EDO-S101 is a hybrid molecule of bendamustine plus vorinostat, new in its class. Our group has previously demonstrated that EDO-S101 is effective in vitro in MM cell lines independently of p53 state, and also in a murine plasmacytoma model where it decreases tumor growth and prolongs survival with respect to bendamustine and/or vorinostat treatment. The objective of this work was to gain further insights into the efficacy of EDO-S101, its mechanism of action and its combination with other drugs used in MM. Methods. The mechanism of action was assessed by western blot, comet assay, immunohistochemistry, and flow cytometry. Homologous recombination (HR) efficiency was calculated using chromosomally integrated green fluorescent protein reporter construct-based assay. The efficacy of different combinations was studied in vitro (HMCLs), in vivo (murine plasmacytoma model CB-17 SCID mice) and ex vivo (cells from patients). Results. In addition to the activity of EDO-S101 in MM cell lines we demonstrated that it was active ex vivo in cells isolated from 7 MM patients, with median IC50 of 5 µM (ranging from 1,8 to 8 µM), some of them previously exposed and resistant to alkylators such as melphalan. Interestingly, EDO-S101 could also overcome alkylators-resistance in vitro, as it was active in melphalan resistant cells (U266-LR7 and RPMI8226-LR5). EDO-S01 was also effective in the presence of factors that confer proliferative advantage to plasma cells, like IL-6, IGF or co-culture with mesenchimal cells hMSC-TERT. Regarding its mechanism of action, we found that the apoptosis induced by EDO-S101 was caspase-independent but calpain-dependent, since PD150606, an inhibitor of this protein could overcome EDO-S101-induced apoptosis, whereas the caspase inhibitor Z-VAD -FMK did not. This data was consistent with the finding that under treatment with EDO-S101, MM1S cells showed AIF (apoptotic inducing factor) translocation from the mitochondria into the nucleus. Interestingly, the release of this pro-apoptotic protein from the mitochondria could be mediated by calpains, as it has been described in literature. We subsequently demonstrated that EDO-S101 causes DNA damage, as revealed by the phosphorylation and subsequent activation of several components of the DNA Damage Response (DDR) such as ATM, H2AX, chk1, chk2 or p53, and the induction of DNA fragmentation, that was detected by the comet assay. EDO-S101 was also found to induce cell cycle arrest in different phases depending on the dose and cell line. It has previously been suggested that DACi may impair DNA repair by inhibiting homologous recombination (HR), a pathway related with genomic instability and progression, very active in MM. Therefore we next evaluated the efficiency of HR using a reported construct that was chromosomally integrated in two MM cell lines, JJN3 and U266. Treatment with EDO-S101 significantly reduced the efficiency of HR in both cell lines, by 50% and 20% of untreated controls respectively. Finally, we tested potential combinations with other antimyeloma agents like lenalidomide and thalidomide; and also with proteasome inhibitors (bortezomib, carfilzomib and oprozomib). EDO-S101 potentiated the activity of all these agents, but the most synergistic combination was that including Bortezomib + Dexamethasone (CI 0,4). This combination was also evaluated in vivo, where it significantly decreased tumor growth and prolonged survival compared to agents in monotherapy and in double combinations. We are currently deepening into the mechanism of action of this combination. Conclusions. EDO S101 is active ex vivo in cells isolated from patients and is able to overcome resistance to alkylators. It induces caspase-independent apoptosis, and cell cycle arrest in MM cell lines. These effects are due to the potent DNA damage which is enhanced by HR impairment induced by the hybrid molecule. Moreover, the combination with bortezomib and dexamethasone is especially attractive to be taken into the clinical setting. Disclosures Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Mateos:Takeda: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Onyx: Consultancy.

scholarly journals Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation.

1997 ◽  
Vol 8 (2) ◽  
pp. 287-301 ◽  
Author(s):  
L Connell-Crowley ◽  
J W Harper ◽  
D W Goodrich
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Retinoblastoma Protein ◽  
Cyclin D ◽  
Cyclin Dependent Kinases ◽  
Cycle Arrest ◽  
Lxcxe Motif ◽  
Hyperphosphorylated Form

The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G1 cyclin-dependent kinases (Cdks) are active, the mechanisms underlying pRb regulation are unknown. In vitro phosphorylation by cyclin D1/Cdk4 leads to inactivation of pRb in a microinjection-based in vivo cell cycle assay. In contrast, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A- or E-type cyclins is not sufficient to inactivate pRb function in this assay, despite extensive phosphorylation and conversion to a slowly migrating "hyperphosphorylated form." The differential effects of phosphorylation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the absence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 phosphorylation in the microinjection assay. This study identifies a residue whose phosphorylation is critical for inactivation of pRb-mediated growth suppression, and it indicates that hyperphosphorylation and inactivation of pRb are not necessarily synonymous.

scholarly journals Peiminine Inhibits Glioblastoma in Vitro and in Vivo Through Cell Cycle Arrest and Autophagic Flux Blocking

2018 ◽  
Vol 51 (4) ◽  
pp. 1566-1583 ◽  
Author(s):  
Boxian Zhao ◽  
Chen Shen ◽  
Zhixing Zheng ◽  
Xiaoxiong Wang ◽  
Wenyang Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Cycle Arrest ◽  
Colony Formation ◽  
Morphological Changes ◽  
Autophagic Flux ◽  
Anticancer Activities ◽  
Cycle Arrest

Background/Aims: Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed. Results: Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo. Conclusion: Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.

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