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

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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Identification of CCND2 As a RUNX1/ETO Target Required for Leukaemic Propagation

Blood ◽

10.1182/blood.v128.22.835.835 ◽

2016 ◽

Vol 128 (22) ◽

pp. 835-835 ◽

Cited By ~ 1

Author(s):

Lynsey McKenzie ◽

Natalia Martinez-Soria ◽

Julia Draper ◽

Sirintra Nakjang ◽

Helen J Blair ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Fusion Protein ◽

Cell Cycle Arrest ◽

Colony Formation ◽

G1 Phase ◽

Cycle Arrest ◽

Gene Sets

Abstract The translocation t(8;21) is the most common chromosomal rearrangement in AML and generates the RUNX1/ETO fusion protein. RUNX1/ETO is required for maintaining the leukaemic phenotype influencing both leukaemic clonogenicity and proliferation and is traditionally thought of as a transcriptional repressor through its actions with histone deacetylases and transcriptional co-repressors. Intriguingly our RNAi experiments have also identified many RUNX1/ETO target genes that are transcriptionally upregulated by this fusion protein. To delve deeper into the involvement of these upregulated genes in RUNX1/ETO driven leukaemia, targeted RNAi screens were performed both in vitro and in vivo, directed at genes found directly bound by RUNX1/ETO and that were found differentially expressed upon RUNX1/ETO knockdown. For the RNAi screens, we used a doxycycline (dox)-inducible lentiviral RNAi library covering each gene with 3 shRNAs. We transduced two t(8;21)-positive AML cell lines, Kasumi-1 and SKNO-1, with this library and performed parallel screens employing colony formation and long-term suspension culture assays in the in vitro arm, and intrafemoral xenotransplantation of immunodeficient NSG mice for the in vivo arm. For comparative purposes, both arms of the RNAi screen were performed in the presence and absence of dox. DNA was isolated throughout both screens and was analysed by Next Generation Sequencing (NGS). Comparison of the changes in level of integrated shRNA coding sequences between dox and no dox groups were made using DESEQ and those genes that are required for RUNX1/ETO driven leukaemia identified. As expected, RUNX1/ETO shRNA constructs diminished upon induction of shRUNX1/ETO expression by dox, therefore implicating RUNX1/ETO dependency in cell survival. Furthermore non-targeting control shRNA (shNTC) levels were unaffected. Out of the many hits identified (which include previously reported hits such as Pontin (RUBVL1), SKP2 and KIT, Cyclin D2 (CCND2) stood out significantly, since levels of shCCND2 were depleted in all dox samples from both arms of the screen. CCND2 is a cell cycle regulator whose activity is dependent on its binding to CDK4/6 in G1 phase. Phosphorylation of Rb (Retinoblastoma), by CDK4/6-CCND2, uncouples Rb from E2F allowing transcription of essential S phase genes. Here we show CCND2 knockdown downregulates both cell proliferation and colony formation in t(8;21) positive cells by causing G1 phase cell cycle arrest via a reduction in Rb phosphorylation, which is a phenotype copied by our RUNX1/ETO knockdown. Moreover, inhibition of CDK4/6-CCND2 by palbociclib (PD-0332991) in t(8;21) positive cells similarly reduces cell proliferation and colony formation via a G1 cell cycle arrest. Interestingly, primary CD34+ human stem/progenitor cells (HSPCs) containing an activated form of KIT (N822K) and the RUNX1/ETO 9a isoform are likewise responsive to palbociclib treatment. In addition to in vitro studies, palbociclib was tested in vivo in two separate experimental models: firstly, mice were intravenously injected with murine CD34+ cKit+ HSPCs expressing the RUNX1/ETO9a isoform and secondly, immunodeficient mice were intrahepatically injected with Kasumi-1. Mice were dosed with palbociclib upon confirmation of engraftment. Engraftment was detected using bioluminescence and survival rates recorded. Both studies showed significant increased survival with palbociclib treatment with the first giving an increased median survival of 59 versus 29 days (p<0.005) and the second giving a median survival of 87 and 67 days for palbociclib treated and control mice (p<0.005), respectively. Finally, Gene Set Enrichment Analysis (GSEA) shows the correlations between palbociclib treatment, CCND2 knockdown and RUNX1/ETO knockdown and shows common gene sets shared between them and other gene sets which include those targeted by MYC and E2F. Interestingly, gene sets that only correlate with knockdown of CCND2 and RUNX1/ETO and evidentially unaffected by CDK4/6 activity will be of interest, in order to target other RUNX1/ETO dependent proteins and pathways alongside of CDK4/6 inhibition. We are currently investigating the use of palbociclib with other drugs in order to develop new drug combinations with reduced toxicity and minimal risk of developing resistance. Disclosures No relevant conflicts of interest to declare.

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

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666181207102037 ◽

2019 ◽

Vol 19 (3) ◽

pp. 365-374 ◽

Cited By ~ 1

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.

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W436, a novel SMART derivative, exhibits anti‐hepatocarcinoma activity by inducing apoptosis and G2/M cell cycle arrest in vitro and in vivo and induces protective autophagy

Journal of Biochemical and Molecular Toxicology ◽

10.1002/jbt.22831 ◽

2021 ◽

Author(s):

Shuai Man ◽

Zhuzhu Wu ◽

Rui Sun ◽

Qi Guan ◽

Zengqiang Li ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

M Cell ◽

Cycle Arrest

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PGM1 Suppresses Colorectal Cancer Cell Migration And Invasion by Regulating PI3K/ AKT Pathway

10.21203/rs.3.rs-944736/v1 ◽

2021 ◽

Author(s):

Zhewen Zheng ◽

Xue Zhang ◽

Jian Bai ◽

Long Long ◽

Di Liu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Cycle ◽

Colony Formation ◽

Tumor Formation ◽

Akt Pathway ◽

Migration And Invasion ◽

Cycle Arrest ◽

Cancer Pathogenesis

Abstract BackgroundPhosphoglucomutase 1(PGM1) is known for its involvement in cancer pathogenesis. However, its biological role in colorectal cancer (CRC) is unknown. Here, we studied the functions and mechanisms of PGM1 in CRC.Methods We verified PGM-1 as a DEG by a comprehensive strategy of the TCGA-COAD dataset mining and computational biology. Relative levels of PGM-1 in CRC tumors and adjoining peritumoral tissue were identified by qRT-PCR, WB, and IHC staining in a tissue microarray. PGM1 functions were analyzed using CCK8, EdU, colony formation, cell cycle, apoptosis, and Transwell migration and invasion assays. The influence of PGM1 was further investigated using tumor formation in vivo.ResultsPGM1 mRNA and protein were both reduced in CRC and the reduction was related to CRC pathology and overall survival. PGM1 knockdown stimulated both proliferation and colony formation, promoting cell cycle arrest and apoptosis while overexpression has opposite effects in CRC cells both in vivo and in vitro. Furthermore, we lined the actions of PGM1 to the PI3K/ AKT pathway. ConclusionWe verified that PGM1 suppresses CRC through the PI3K/ AKT pathway. These results suggest the potential for targeting PGM1 in CRC therapies.

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Menadione reduces CDC25B expression and promotes tumor shrinkage in gastric cancer

Therapeutic Advances in Gastroenterology ◽

10.1177/1756284819895435 ◽

2020 ◽

Vol 13 ◽

pp. 175628481989543

Author(s):

Amanda Braga Bona ◽

Danielle Queiroz Calcagno ◽

Helem Ferreira Ribeiro ◽

José Augusto Pereira Carneiro Muniz ◽

Giovanny Rebouças Pinto ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Specific Inhibitor ◽

Gastric Carcinogenesis ◽

In Vivo Experiments ◽

Cycle Arrest

Background: Gastric cancer is one of the most incident types of cancer worldwide and presents high mortality rates and poor prognosis. MYC oncogene overexpression is a key event in gastric carcinogenesis and it is known that its protein positively regulates CDC25B expression which, in turn, plays an essential role in the cell division cycle progression. Menadione is a synthetic form of vitamin K that acts as a specific inhibitor of the CDC25 family of phosphatases. Methods: To better understand the menadione mechanism of action in gastric cancer, we evaluated its molecular and cellular effects in cell lines and in Sapajus apella, nonhuman primates from the new world which had gastric carcinogenesis induced by N-Methyl-N-nitrosourea. We tested CDC25B expression by western blot and RT-qPCR. In-vitro assays include proliferation, migration, invasion and flow cytometry to analyze cell cycle arrest. In in-vivo experiments, in addition to the expression analyses, we followed the preneoplastic lesions and the tumor progression by ultrasonography, endoscopy, biopsies, histopathology and immunohistochemistry. Results: Our tests demonstrated menadione reducing CDC25B expression in vivo and in vitro. It was able to reduce migration, invasion and proliferation rates, and induce cell cycle arrest in gastric cancer cell lines. Moreover, our in-vivo experiments demonstrated menadione inhibiting tumor development and progression. Conclusions: We suggest this compound may be an important ally of chemotherapeutics in the treatment of gastric cancer. In addition, CDC25B has proven to be an effective target for investigation and development of new therapeutic strategies for this malignancy.

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Bound polyphenol extracted from jujube pulp triggers mitochondria-mediated apoptosis and cell cycle arrest of HepG2 cell in vitro and in vivo

Journal of Functional Foods ◽

10.1016/j.jff.2018.12.017 ◽

2019 ◽

Vol 53 ◽

pp. 187-196 ◽

Cited By ~ 2

Author(s):

Shuhua Shan ◽

Yue Xie ◽

Huiling Zhao ◽

Jinping Niu ◽

Sheng Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Hepg2 Cell ◽

Cycle Arrest

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Growth Suppression by Acute PromyelocyticLeukemia-Associated Protein PLZF Is Mediated by Repression ofc-mycExpression

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.9375-9388.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 9375-9388 ◽

Cited By ~ 99

Author(s):

Melanie J. McConnell ◽

Nathalie Chevallier ◽

Windy Berkofsky-Fessler ◽

Jena M. Giltnane ◽

Rupal B. Malani ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Cycle Arrest ◽

Target Genes ◽

Ectopic Expression ◽

Growth Suppression ◽

Quiescent State ◽

Cycle Arrest

ABSTRACT The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control.

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