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Naringin, a bioflavonoid compound from grapefruit or citrus, exerts anticancer activities on cervical, thyroid, colon, brain, liver, lung, thyroid, and breast cancers. The present investigation addressed exploring the anticancer effects of naringin on nasopharyngeal carcinoma (NPC) cells. Naringin exhibits a cytotoxic effect on NPC‐TW 039 and NPC‐TW 076 cells with IC50 372/328 and 394/307 μM for 24 or 48 h, respectively, while causing little toxicity toward normal gingival epithelial (SG) cells (>500/500 μM). We established that naringin triggered G1 arrest is achieved by suppressing cyclin D1, cyclin A, and CDK2, and upregulating p21 protein in NPC cells. Exposure of NPC cells to naringin caused a series of events leading to apoptosis including morphology change (cell shrinkage and membrane blebbing) and chromatin condensation. Annexin V and PI staining indicated that naringin treatment promotes necrosis and late apoptosis in NPC cells. DiOC6 staining showed a decline in the mitochondrial membrane potential by naringin treatment, which was followed with cytochrome c release, Apaf‐1/caspase‐9/‐3 activation, PARP cleavage, and EndoG expression in NPC cells. Naringin upregulated proapoptotic Bax and decreased antiapoptotic Bcl‐xL expression, and dysregulated Bax/Bcl‐xL ratio in NPC cells. Notably, naringin enhanced death receptor‐related t‐Bid expression. Furthermore, an increased Ca2+ release by naringin treatment which instigated endoplasmic reticulum stress‐associated apoptosis through increased IRE1, ATF‐6, GRP78, GADD153, and caspase‐12 expression in NPC cells. In addition, naringin triggers ROS production, and inhibition of naringin‐induced ROS generation by antioxidant N‐acetylcysteine resulted in the prevention of G1 arrest and apoptosis in NPC cells. Naringin‐induced ROS‐mediated G1 arrest and mitochondrial‐, death receptor‐, and endoplasmic reticulum stress–mediated apoptosis may be a promising strategy for treating NPC. [ABSTRACT FROM AUTHOR]

Simple Summary: Cell cycle synchronization method could be used to improve the diagnostic sensitivity of oncologic FDG positron emission tomography. Background: We investigated whether cell cycle synchronization induced by the T-type calcium channel inhibitor mibefradil could increase tumoral 2-[18F] fluoro-2-deoxy-d-glucose (FDG) uptake in vitro and in vivo. Methods: Human prostate cancer cells (PC-3) were treated with 10 μM mibefradil for 24, 48, and 72 h to induce G1 arrest. Cell cycle distribution was analyzed at 0, 4, 8, 12, 15, 18, and 24 h after mibefradil withdrawal. Cellular uptake was measured after incubating cells with [3H] Deoxy-d-Glucose (DDG) for 1 h at the same time points used in the cell cycle analysis. The correlation between [3H] DDG uptake and each cell cycle phase was evaluated in the early (0–12 h) and late phases (15–24 h) of synchronization. In vivo FDG PET imaging was performed in PC-3-bearing mice at baseline, 24 h, and 48 h after mibefradil treatment. Results: The G0/G1 fraction of PC-3 cells was significantly increased from 33.1% ± 0.2% to 60.9% ± 0.8% after 24 h mibefradil treatment, whereas the S and G2/M fractions were decreased from 36.3% ± 1.4% to 23.2% ± 1.1% and from 29.7% ± 1.3% to 14.9% ± 0.9%, respectively, which were similar to the results by serum starvation. Mibefradil treatment for 24, 48, and 72 h increased the number of cells in S phase at 18–24 h after withdrawal; however, only the 72 h treatment increased [3H] DDG uptake (145.8 ± 5.8% of control at 24 h after withdrawal). [3H] DDG uptake was positively correlated with the size of the S phase fraction and negatively correlated with the size of the G0/G1 fraction in the late phase of synchronization. DDG uptake was significantly increased by mibefradil-induced cell cycle synchronization and correlated with the sizes of cell cycle fractions. In vivo FDG PET imaging also demonstrated a significant increase in tumor uptake after mibefradil treatment. Quantified tumor FDG uptake (%ID/g) increased from 4.13 ± 2.10 to 4.7 ± 2.16 at 24 h, and 5.95 ± 2.57 at 48 h (p < 0.05). Conclusion: Cell cycle synchronization could be used to increase the diagnostic sensitivity of clinical FDG positron emission tomography. [ABSTRACT FROM AUTHOR]

Simple Summary: The cell cycle is divided in four phases, the G1 phase for growth in cell size and increased protein biosynthesis, the S phase for the synthesis and replication of DNA, and the G2 phase for preparing the cell for the M phase, the phase of cell division. Cell cycle inhibitors control progression through the cell cycle. The cell cycle inhibitor p21 arrests cells in the G1 phase correlating with a prolonged protein production phase. This effect could be used to increase the production of biotherapeutic proteins. Here, we applied an optogenetic approach to control the function of p21. Optogenetics is an emerging field within synthetic biology and based on genetically encoded light-sensitive elements derived from plants, fungi or bacteria. Optogenetic tools can be used to control biological functions such as signaling pathways, metabolic pathways or gene expression via light with less side effects than when using chemical inducers. In this study, we designed and applied light switches to control the subcellular localization and thereby the function of p21via light. The stimulation of light-regulated p21 increased the number of cells arrested in the G1 phase correlating with the increased expression of a reporter protein. Implementation of this system could be used to optimize the production of biotherapeutic protein. The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 plays a critical role in cancer pathogenesis, and regulation of p53 expression is essential for maintaining normal cell growth. UBE4B is an E3/E4 ubiquitin ligase involved in a negative-feedback loop with p53. UBE4B is required for Hdm2-mediated p53 polyubiquitination and degradation. Thus, targeting the p53-UBE4B interactions is a promising anticancer strategy for cancer therapy. In this study, we confirm that while the UBE4B U box does not bind to p53, it is essential for the degradation of p53 and acts in a dominant-negative manner, thereby stabilizing p53. C-terminal UBE4B mutants lose their ability to degrade p53. Notably, we identified one SWIB/Hdm2 motif of UBE4B that is vital for p53 binding. Furthermore, the novel UBE4B peptide activates p53 functions, including p53-dependent transactivation and growth inhibition, by blocking the p53-UBE4B interactions. Our findings indicate that targeting the p53-UBE4B interaction presents a novel approach for p53 activation therapy in cancer.

Colorectal cancer (CRC) is recognized as the third most common malignancy and the second most deadly in highly developed countries. Although the treatment of CRC has improved in the past decade, the mortality rate of CRC is still increasing. Amentoflavone, one of the flavonoids detected in medical plants, is reported to possess potential anticancer properties in various cancers. However, its role in CRC has not been studied. This study aimed to investigate the role and underlying mechanism of amentoflavone on CRC in vitro and in vivo. We identified the cytotoxicity, apoptosis effect, cell cycle alteration, DNA damage induction and tumor progression inhibition of amentoflavone in HT‐29 model by using MTT assay, flow cytometry, immunofluorescence (IF) staining, Western blotting and animal experiments. Amentoflavone induced cytotoxicity is caused by triggering G1 arrest, DNA damage and apoptosis in HT‐29 cells. The expression of cyclin D1, CDK4 and CDK6 was decreased by amentoflavone; in contrast, the phosphorylation of ATM and CHK2 and the expression of p21 and p27 were increased. The apoptosis induction of amentoflavone in CRC is not only caspase‐dependent but also increases EndoG and AIF nuclear translocation in a caspase‐independent manner. Importantly, the apoptosis induction of amentoflavone is not affected by the activity of p53 in CRC. Amentoflavone suppressed the progression of CRC by initiating G1 arrest and ATM/CHK2‐mediated DNA damage‐responsive, caspase‐dependent/independent apoptotic effects. We uncovered a novel tumor‐inhibitory role of amentoflavone in CRC that is not associated with p53 activity, which may serve as a potential treatment for CRC. [ABSTRACT FROM AUTHOR]

The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Modulation of the host cell cycle has emerged as a common theme among the pathways regulated by bacterial pathogens, arguably to promote host cell colonization. However, in most cases the exact benefit ensuing from such interference to the infection process remains unclear. Previously, we have shown that Salmonella actively induces G2/M arrest of host cells, and that infection is severely inhibited in cells arrested in G1. In this study, we demonstrate that Salmonella vacuolar replication is inhibited in host cells blocked in G1, whereas the cytosolic replication of the closely related pathogen Shigella is not affected. Mechanistically, we show that cells arrested in G1, but not cells arrested in G2, present dysregulated endolysosomal trafficking, displaying an abnormal accumulation of vesicles positive for late endosomal and lysosomal markers. In addition, the macroautophagic/autophagic flux and degradative lysosomal function are strongly impaired. This endolysosomal trafficking dysregulation results in sustained activation of the SPI-1 type III secretion system and lack of vacuole repair by the autophagy pathway, ultimately compromising the maturation and integrity of the Salmonella-containing vacuole. As such, Salmonella is released in the host cytosol. Collectively, our findings demonstrate that the modulation of the host cell cycle occurring during Salmonella infection is related to a disparity in the permissivity of cells arrested in G1 and G2/M, due to their intrinsic characteristics. Abbreviations: CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CDK4-CDK6i: CDK4-CDK6 inhibitor IV; cfu: colony-forming units; CHQ: chloroquine; DMSO: dimethyl sulfoxide; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; hpi: hours post-infection; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; RAB7: RAB7, member RAS oncogene family; SCV: Salmonella-containing vacuole; SPI-1: Salmonella pathogenicity island-1; SPI-2: Salmonella pathogenicity island-2; TFEB: transcription factor EB; T3SS: type III secretion system [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the most frequently occurring liver malignancy in Asia. Glycyrrhizic acid is known to reduce the risk of HCC formation in patients with chronic hepatitis C. To identify whether glycyrrhizic acid may play a role in anti‐HCC therapy as an adjuvant is important. However, the inhibitory effect of glycyrrhizic acid on cell cycle progression in HCC cells and the mechanism of such have not been fully elucidated. This study used the comet assay, cell cycle analysis, immunofluorescence staining, the TUNEL assay, and Western blotting to identify the anti‐HCC role of glycyrrhizic acid. Glycyrrhizic acid may induce DNA damage, apoptosis, activation of ATM, and expression of p21, and p27 in HCC cells. In addition, glycyrrhizic acid may also induce G1 phase arrest and suppress NF‐κB‐mediated Cyclin D1 expression. DNA damage and NF‐κB inactivation may be associated with glycyrrhizic acid‐induced G1 phase arrest in HCC cells. [ABSTRACT FROM AUTHOR]

Objective: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. Methods: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. Results: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). Conclusion: KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells. [ABSTRACT FROM AUTHOR]

Background. Triple-negative breast cancer (TNBC) responds poorly to the available drugs; thus, the mortality rate associated with TNBC remains high. 7-α-Hydroxyfrullanolide (7HF) possesses anticancer properties and arrests cells in the G2/M-phase via modulation of several proteins involved in the G2/M-phase transition, as well as the mitotic checkpoint in MDA-MB-468 (TNBC) cells. Microtubules (MTs) dynamically regulate cell division in the G2/M phase and are related to cancer cell stress response. However, antimitotic drug cytotoxicity to multiple cancer resistance developed in response to drugs are obstacles faced to date. Here, the activity and mechanism via which 7HF controls MTs dynamics was investigated in MDA-MB-468 cells. Methods. 7HF uptake by MDA-MB-468 cells was assessed using spectrophotometry. The drug-like properties of 7HF were predicted using the Swiss-absorption, distribution, metabolism, and excretion (ADME) webtool. Then, the effect of 7HF treatment (6, 12, and 24 mM) on the dynamic arrangement of MTs was assessed for 1, 12, and 24 h using indirect immunofluorescence. Polymerization of α- and β-tubulin was assessed using different 7HF concentrations in a cell-free system for 1 h. Cell proliferation assay with bromodeoxyuridine plus propidium iodide staining and flow cytometry was performed at different 7HF concentrations and time points. The mechanism of action was assessed by detecting the expression of proteins, including Bub3, cyclin B1, p-Cdk1 (Tyr15), Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139), using western blotting. Molecular docking was used to predict the molecular interactions between 7HF and tubulins in MTs. Results. We observed that 7HF was able to enter the MDA-MB-468 cells. The ADME webtool analysis predicted that it possesses the high passive permeation and gastrointestinal absorption properties of drugs. Various concentrations of 7HF disrupted the dynamic arrangement of spindle MTs by causing radial spindle array shrinkage and expansion of fibrous spindle density and radial array lengths in a time-dependent manner. 7HF reduced polymerization of α-, β-tubulin in dose-dependent manner. 7HF also triggered DNA damage response by inducing G2/M and G1 phase arrests in a concentration and time-dependent manner, which occurred due to the upregulation of Bub3, Chk1, p-Chk1 (Ser345), p-Cdk1 (Tyr15), and cyclin B1. According to molecular docking analysis, 7HF preferred to bind to β-tubulin over α-tubulin. The lactone, ketone, and hydroxyl groups of 7HF supported the 7HF-tubulin interactions. Hydrogen bonding with a hydrocarbon ring and salt bridge attractive forces were responsible for the binding versatility of 7HF. Conclusions. This is the first study to investigate the molecular mechanism, MTs interacting sites, and the internalization and drug-like properties of 7HF in TNBC cells. The findings will be useful for developing 7HF-based treatment for patients with TNBC. [ABSTRACT FROM AUTHOR]

Lung cancer is a leading cause of cancer-related mortality and remains one of the most poorly prognosed disease worldwide. Therefore, it is necessary to identify novel molecular markers with potential therapeutic effects. Recent findings have suggested that dual-specificity tyrosine-regulated kinase 2 (DYRK2) plays a tumor suppressive role in colorectal, breast, and hepatic cancers; however, its effect and mechanism in lung cancer remain poorly understood. Therefore, this study aimed to investigate the tumor-suppressive role and molecular mechanism of DYRK2 in lung adenocarcinoma (LUAD) by in vitro experiments and xenograft models. The evaluation of DYRK2 expression was carried out using lung cancer cell lines and normal bronchial epithelial cells. Overexpression of DYRK2 was induced by an adenovirus vector, and cell proliferation was assessed through MTS assay and Colony Formation Assay. Cell cycle analysis was performed using flow cytometry. Additionally, proliferative capacity was evaluated in a xenograft model by subcutaneously implanting A549 cells into SCID mice (C·B17/Icr-scidjcl-scid/scid). Immunoblotting assays showed that DYRK2 was downregulated in most LUAD cell lines. DYRK2 overexpression using adenovirus vectors significantly suppressed cell proliferation compared with that in the control group. Additionally, DYRK2 overexpression suppressed tumor growth in a murine subcutaneous xenograft model. Mechanistically, DYRK2 overexpression inhibited the proliferation of LUAD cells via p21-mediated G1 arrest, which was contingent on p53. Taken together, these findings suggest that DYRK2 may serve as potential prognostic biomarker and therapeutic target for LUAD. • DYRK2 overexpression suppresses lung adenocarcinoma via p53 and p21 upregulation. • DYRK2 induces G1 arrest in lung adenocarcinoma cell lines. • DYRK2 expression may be a prognostic factor in lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Naringin, a bioflavonoid compound from grapefruit or citrus, exerts anticancer activities on cervical, thyroid, colon, brain, liver, lung, thyroid, and breast cancers. The present investigation addressed exploring the anticancer effects of naringin on nasopharyngeal carcinoma (NPC) cells. Naringin exhibits a cytotoxic effect on NPC-TW 039 and NPC-TW 076 cells with IC 50 372/328 and 394/307 μM for 24 or 48 h, respectively, while causing little toxicity toward normal gingival epithelial (SG) cells (>500/500 μM). We established that naringin triggered G 1 arrest is achieved by suppressing cyclin D1, cyclin A, and CDK2, and upregulating p21 protein in NPC cells. Exposure of NPC cells to naringin caused a series of events leading to apoptosis including morphology change (cell shrinkage and membrane blebbing) and chromatin condensation. Annexin V and PI staining indicated that naringin treatment promotes necrosis and late apoptosis in NPC cells. DiOC 6 staining showed a decline in the mitochondrial membrane potential by naringin treatment, which was followed with cytochrome c release, Apaf-1/caspase-9/-3 activation, PARP cleavage, and EndoG expression in NPC cells. Naringin upregulated proapoptotic Bax and decreased antiapoptotic Bcl-xL expression, and dysregulated Bax/Bcl-xL ratio in NPC cells. Notably, naringin enhanced death receptor-related t-Bid expression. Furthermore, an increased Ca 2+ release by naringin treatment which instigated endoplasmic reticulum stress-associated apoptosis through increased IRE1, ATF-6, GRP78, GADD153, and caspase-12 expression in NPC cells. In addition, naringin triggers ROS production, and inhibition of naringin-induced ROS generation by antioxidant N-acetylcysteine resulted in the prevention of G 1 arrest and apoptosis in NPC cells. Naringin-induced ROS-mediated G 1 arrest and mitochondrial-, death receptor-, and endoplasmic reticulum stress-mediated apoptosis may be a promising strategy for treating NPC., (© 2024 Wiley Periodicals LLC.)

The main biological function of the tumor suppressor p53 is to control cell cycle arrest and apoptosis. Among the p53 target genes, p21 has been identified as a key player in p53-mediated G1 arrest, while Killin, via its high DNA binding affinity, has been implicated in S and G2/M arrest. However, whether Killin is involved in G1 arrest remains unclear. This research aimed to explore the role of Killin in p53-mediated G1 arrest. Knockout of killin in human colorectal cells led to a dramatic decrease in p53-mediated G1 arrest upon DNA damage. Moreover, double knockout of killin and p21 completely abolished G1 arrest, similar to that of p53 knockout cells. We further showed that Killin could upregulate p21 protein expression independent of p53 via ubiquitination pathways. Immunoprecipitation studies indicated that Killin may directly bind to proteasome subunits, thereby disrupting proteasomal degradation of p21. Together, these results demonstrate that Killin is involved in multiple cell cycle checkpoint controls, including p53-mediated G1 arrest. [ABSTRACT FROM AUTHOR]

Abstract Background Genomic instability is a hallmark of cancer. The G1 checkpoint allows cells to repair damaged DNA that may lead to genomic instability. The high-risk human papillomavirus (HPV) E7 gene can abrogate the G1 checkpoint, yet the mechanism is still not fully understood. Our recent study showed that WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein 1) plays a role in regulating G1 checkpoint of E7 expressing cells. In this study, we explored the mechanism by which WDHD1 regulates G1 checkpoint in HPV E7 expressing cells. Methods NIKS and RPE1 derived cell lines were used. Real-time PCR, Rescue experiment, FACS and BrdU labeling experiments were performed to examine role of GCN5 in G1 checkpoint abrogation in HPV-16 E7 expressing cells. Results In this study, we observed that WDHD1 facilitates G1 checkpoint abrogation by modulating GCN5 in HPV E7 expressing cells. Notably, depletion of WDHD1 caused G1 arrest while overexpression of GCN5 rescued the inhibitory effects of WDHD1 knockdown on G1/S progression. Furthermore, siWDHD1 significantly decreased cell cycle proliferation and DNA synthesis that was correlated with Akt phosphorylation (p-Akt), which was reversed by GCN5 overexpression in HPV E7 expressing cells. Conclusions In summary, our data identified a WDHD1/GCN5/Akt pathway leading to the abrogation of G1 checkpoint in the presence of damaged DNA, which may cause genomic instability and eventually HPV induced tumorigenesis.

Xudong Ma,1 Jing Luo,1 Yi Zhang,2 Dali Sun,1 Yueying Lin1 1Gastrointestinal Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming City, Yunnan Province 650031, People’s Republic of China; 2The General Surgery Department, The Teng-Chong People’s Hospital, Tengchong City, Yunan Province, 679100, People’s Republic of ChinaCorrespondence: Yueying Lin Email us5010@163.comIntroduction: This study aimed to investigate the role of lncRNA MCM3AP-AS1 in colorectal cancer (CRC).Methods: Paired tumor and non-tumor tissues were collected from 60 CRC patients. Expression of MCM3AP-AS1 was determined by RT-qPCR. Overexpression of MCM3AP-AS1, miR-545, and CDK4 in CRC cells was achieved to explore the interactions between them. Cell cycle assay was performed to analyze the roles of MCM3AP-AS1, miR-545, and CDK4 in regulating the cell cycle progression of CRC cells.Results: We found that MCM3AP-AS1 was upregulated in CRC and its high expression levels predicted poor survival of CRC patients. MCM3AP-AS1 is predicted to interact with miR-545. In CRC cells, overexpression of MCM3AP-AS1 and miR-545 was achieved, while their overexpression did not affect the expression of each other. Instead, overexpression of MCM3AP-AS1 led to the increased expression levels of CDK4, which is a downstream target of miR-545. Cell cycle analysis showed that overexpression of MCM3AP-AS1 and CDK4 suppressed G1 arrest induced by miR-545. In addition, overexpression of MCM3AP-AS1 reduced the enhancing effects of overexpressing miR-545 on cell cycle progression.Conclusion: MCM3AP-AS1 may upregulate CDK4 by sponging miR-545 to induce G1 arrest in CRC cells.Keywords: MCM3AP-AS1, colorectal cancer, miR-545, CDK4, G1 arrest

Ying Zhang,1 Yan Miao,1 Muhe Shang,1 Mxin Liu,1 Ran Liu,1 Ench Pan,2 Yuepu Pu,1 Lihong Yin11Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, People’s Republic of China; 2Huaian Center for Disease Control and Prevention, Huaian 223001, People’s Republic of ChinaBackground: Esophageal squamous cell carcinoma (ESCC) is the fourth most common cause of cancer death in China. Long noncoding RNAs have emerged as critical regulators in cancer. Long intergenic noncoding RNA-p21, a kind of Long noncoding RNAs, LincRNA-p21 have been discussed dysregulated in several cancers, but its role in ESCC remains unknown. This study investigated the role of LincRNA-p21 in ESCC.Materials and methods: The LincRNA-p21 expression level and its association with esophageal cancer was determined in 64 tumor tissues of esophageal squamous cell carcinoma patients and cells using quantitative real-time reverse transcription PCR. Fluorescence in situ hybridization of single-RNA molecular probes was used to determine subcellular localization of LincRNA-p21. CCK8 and EdU assays were used for proliferation assay, flow cytometry was performed for apoptosis and cell-cycle distribution, and 24-well Mill cell chamber was made for measuring the abilities of migration and invasion after transfected with lentivirus-expressing LincRNA-p21 in EC109 cells. Then, quantitative real-time reverse transcription PCR and Western blot detected the expression of p21. Further, UC2288, an inhibitor of p21, was used to decrease the level of p21, and flow cytometry was used to detect cell cycle. Finally, screening for differential pathways from microarray analysis and expression of p53 and cyclin D were detected by Western blot.Results: LincRNA-p21 expression level was remarkably lower in tumor tissues versus nontumor tissues and lower in EC109 cells versus Het-1A cells. Statistical analysis found that LincRNA-p21 might enhance the risk of ESCC. We observed that LincRNA-p21 was expressed both in the nucleus and cytoplasm, and a larger proportion of LincRNA-p21 was observed in the cytoplasm. The results demonstrated that upregulating the expression of LincRNA-p21 could inhibit cell proliferation, migration, invasion, and the transition of cell cycle from G1 and promoted apoptosis of EC109. Then, we found that LincRNA-p21 promotes the expression of p21. Decreasing the level of p21 revealed that cell-cycle arrest was restored. Pathway analysis found p53 pathway was downregulated, and upregulation of LincRNA-p21 inhibited the expression of cyclin D.Conclusion: Our study suggests that LincRNA-p21 plays as a tumor inhibitor in ESCC development and LincRNA-p21 might induce G1 arrest through p53 signal pathway.Keywords: esophageal squamous-cell carcinoma, LincRNA-p21, G1 arrest, p53 pathway, Fluorescence in Situ Hybridization

Summary: Most solid tumors are aneuploid, and p53 has been implicated as the guardian of the euploid genome. Previous experiments using human cell lines showed that aneuploidy induction leads to p53 accumulation and p21-mediated G1 cell cycle arrest. We find that adherent 2-dimensional (2D) cultures of human immortalized or cancer cell lines activate p53 upon aneuploidy induction, whereas suspension cultures of a human lymphoid cell line undergo a p53-independent cell cycle arrest. Surprisingly, 3D human and mouse organotypic cultures from neural, intestinal, or mammary epithelial tissues do not activate p53 or arrest in G1 following aneuploidy induction. p53-deficient colon organoids have increased aneuploidy and frequent lagging chromosomes and multipolar spindles during mitosis. These data suggest that p53 may not act as a universal surveillance factor restricting the proliferation of aneuploid cells but instead helps directly or indirectly ensure faithful chromosome transmission likely by preventing polyploidization and influencing spindle mechanics.

A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence‐specific repressors. The final division that takes place post‐DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin‐dependent, long‐range chromatin interactions lead to genome‐wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast. Take Away: The transition to quiescence begins before glucose limitation.Abrupt glucose starvation does not trigger quiescence.Quiescence‐specific repressors recruit Rpd3L histone deacetylase.Histones undergo massive ubiquitylation, methylation and acetylation changes.Quiescent chromatin compaction is conserved in yeast and human cells. [ABSTRACT FROM AUTHOR]

Background/Aim: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and the third leading cause of cancer death worldwide. Although multiple chemotherapies options are available for HCC, chemo-induced toxicity is inevitable during clinical treatment. Therefore, identifying possible adjuvant agents with both liver-protective and antitumor effects is critical. Herbal medicines have chemopreventive and anti-HCC effect, such as Juzen taiho-to and Sho-saiko-to. Astragaloside IV is a compound extracted from the Chinese medical herb Astragalus membranaceus (Fisch.) Bge. with liver protection potential. However, whether astragaloside IV may also possess tumor-inhibitory capability and its underlying mechanism is remaining unknown. Materials and Methods: Viability analysis, cell-cycle analysis, apoptosis analysis, western blotting analysis and invasion trans-well assay were performed to identify tumor-inhibitory potential of astragaloside IV on HCC cells (SK-Hep1 and Hep3B cells). Results: We found that astragaloside IV may induce cytotoxicity and extrinsic/intrinsic apoptosis effect, but also trigger G1 arrest in HCC cells. The expression of anti-apoptotic proteins of HCC were all reduced by astragaloside IV. Additionally, astragaloside IV also suppressed HCC cell invasion ability. Conclusion: Astragaloside IV effectively suppressed HCC cell proliferation, invasion and anti-apoptosis in vitro. [ABSTRACT FROM AUTHOR]

Aims: Long non-coding RNA (lncRNA) NR2F2 antisense RNA 1 (NR2F2-AS1) is an oncogenic lncRNA in lung cancer. This study aimed to investigate the role of NR2F2-AS1 in colorectal cancer (CRC).Methods: Tissue specimens were obtained from 63 CRC patients, and gene expression was analyzed by qPCR and western blot. Overexpression was performed to analyze gene interactions. A 5-year follow-up was carried out to perform survival analysis. Cell cycle progression and proliferation were analyzed by cell cycle assay and CCK-8 assay, respectively.Results: We found that NR2F2-AS1 and cyclin-dependent kinase 6 (CDK6) were both upregulated in CRC and were positively correlated. NR2F2-AS1 siRNA silencing led to downregulated CDK6 and induced Gap 1 (G1) arrest of CRC cells. CDK6 overexpression rescued G1 arrest caused by NR2F2-AS1 siRNA silencing. High expression levels of NR2F2-AS1 were closely correlated with low overall 5-year survival rate. NR2F2-AS1 siRNA silencing led to decreased rate of CRC cell proliferation. CDK6 overexpression attenuated the effects of NR2F2-AS1 siRNA silencing on cancer cell proliferation.Conclusions: Downregulation of NR2F2-AS1 induces G1 arrest of CRC cells by downregulating CDK6. [ABSTRACT FROM AUTHOR]

Purpose: 6-Gingerol, a major biochemical and pharmacological active ingredient of ginger, has shown anti-inflammatory and antitumor activities against various cancers. Searching for natural products with fewer side effects for developing adjunctive therapeutic options is necessary.Methods: The effects of 6-gingerol on proliferation, colony formation, and cell cycle in RCC cells were detected by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and propidium iodide (PI) staining, respectively. Western blotting, an immunofluorescence assay, and immunohistochemical staining were performed to assess the expression of relevant proteins. A subcutaneous tumor model was set up to investigate the 6-gingerol effects on tumor growth in vivo, and the pharmacokinetics of 6-gingerol in mice were detected by LC/MS assays.Results: 6-Gingerol treatment exerted time- and dose-dependent inhibition of the growth and colony formation of ACHN, 786-O, and 769-P cells, leading to a concomitant induction of cell-cycle G1-phase arrest and decrease in Ki-67 expression in the cell nucleus. Western-blotting results showed that 6-gingerol reduces phosphorylation of protein kinase B (AKT) Ser 473, cyclin-dependent kinases (CDK4), and cyclin D1 and, meanwhile, increases glycogen synthase kinase (GSK 3β) protein amount. Furthermore, the efficacy of 6-gingerol was demonstrated in an in vivo murine model of 786-O.Conclusion: The above results indicate that 6-gingerol can induce cell-cycle arrest and cell-growth inhibition through the AKT-GSK 3β-cyclin D1 signaling pathway in vitro and in vivo, suggesting that 6-gingerol should be useful for renal-cell carcinoma treatment. [ABSTRACT FROM AUTHOR]

Background: It is known that LINC00974 is an oncogenic long noncoding RNA in liver cancer. Results: The authors observed in this study that LINC00974 was upregulated in gastric cancer (GC) and positively correlated with CDK6. Survival analysis showed that high levels of LINC00974 and CDK6 predicted poor survival. In GC tissues, LINC00974 and CDK6 were positively correlated. In GC cells, LINC00974 overexpression led to upregulated, whereas LINC00974 siRNA silencing led to downregulated CDK6. Analysis of cell cycle progression and proliferation showed that LINC00974 and CDK6 overexpression promoted and siRNA silencing inhibited G1-S transition and cell proliferation. Conclusion: Therefore, LINC00974 upregulates CDK6 to promote cell cycle progression in GC. [ABSTRACT FROM AUTHOR]

In Chinese medicine, herbal medicine is commonly used to treat individuals suffering from many types of diseases. We thus expected that some herbal medicines would contain promising compounds for cancer chemotherapy. Indeed, we found that Sanguisorba officinalis extracts strongly inhibit the growth of B16F10 melanoma cells, and we identified ellagic acid (EA) as the responsible ingredient. B16F10 cells treated with EA exhibited strong G1 arrest accompanied by accumulation of p53, followed by inactivation of AKT. Addition of a PTEN inhibitor, but not a p53 inhibitor, abrogated the EA‐induced AKT inactivation and G1 arrest. The PTEN inhibitor also diminished EA‐induced p53 accumulation. Furthermore, EA apparently increased the protein phosphatase activity of PTEN, as demonstrated by the reduced phosphorylation level of FAK, a protein substrate of PTEN. Furthermore, an in vitro PTEN phosphatase assay on PIP3 showed the direct modulation of PTEN activity by EA. These results suggest that EA functions as an allosteric modulator of PTEN, enhancing its protein phosphatase activity while inhibiting its lipid phosphatase activity. It is notable that a combination of EA and cisplatin, a widely used chemotherapy agent, dramatically enhanced cell death in B16F10 cells, suggesting a promising strategy in chemotherapy. [ABSTRACT FROM AUTHOR]

Though Morusin isolated from the root of Morus alba was known to have antioxidant, anti-inflammatory, antiangiogenic, antimigratory, and apoptotic effects, the underlying antitumor effect of Morusin is not fully understood on the glycolysis of liver cancers. Hence, in the current study, the antitumor mechanism of Morusin was explored in Hep3B and Huh7 hepatocellular carcninomas (HCC) in association with glycolysis and G1 arrest. Herein, Morusin significantly reduced the viability and the number of colonies in Hep3B and Huh7 cells. Moreover, Morusin significantly increased G1 arrest, attenuated the expression of cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase 6 (CDK6) and upregulated p21 and p27 in Hep3B and Huh7 cells. Interestingly, Morusin significantly activated phosphorylation of the adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) but attenuated the expression of the p-mammalian target of protein kinase B (AKT), rapamycin (mTOR), c-Myc, hexokinase 2(HK2), pyruvate kinases type M2 (PKM2), and lactate dehydrogenase (LDH) in Hep3B and Huh7 cells. Consistently, Morusin suppressed lactate, glucose, and adenosine triphosphate (ATP) in Hep3B and Huh7 cells. Conversely, the AMPK inhibitor compound C reduced the ability of Morusin to activate AMPK and attenuate the expression of p-mTOR, HK2, PKM2, and LDH-A and suppressed G1 arrest induced by Morusin in Hep3B cells. Overall, these findings suggest that Morusin exerts an antitumor effect in HCCs via AMPK mediated G1 arrest and antiglycolysis as a potent dietary anticancer candidate.

Background/Aims: CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types; however, its prognostic impact and role in cellular processes, particularly in thyroid carcinoma, are still unclear. Methods: To elucidate the prognostic impact of CRIP1, we analyzed tissues from 58 primary invasive thyroid carcinomas using immunohistochemistry. Western blotting was performed to investigate CRIP1 protein expression in the thyrocyte cell line Nthy-ori 3-1 and four different thyroid carcinoma cell lines, K1, TPC-1, TT, and SW579. Endogenous expression of CRIP1 was suppressed using a siRNA (si-CRIP1). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate cell viability. Flow cytometric analysis was used to detect cell cycle progression and cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion were detected using the transwell assay. Results: The immunohistochemistry results showed that CRIP1 was overexpressed in thyroid carcinoma. CRIP1 expression was associated with tumor size, TNM stage, and lymphatic metastasis, but not with age, gender, and tumor location. In addition, the expression of CRIP1 in K1, TPC-1, TT, and SW529 cells was higher than that in the Nthy-ori 3-1 cells. The highest expression was observed in the SW579 and TT cells. Furthermore, silencing CRIP1 inhibited the proliferation, migration, and invasion of thyroid carcinoma cell lines SW579 and TT. We also found that silencing CRIP1 induced G1 arrest and apoptosis of thyroid carcinoma cell lines SW579 and TT. Conclusion: In conclusion, CRIP1 acts as an oncogene in the cell proliferation, migration, and invasion processes of thyroid carcinoma. CRIP1 may serve well as an independent prognostic marker with significant predictive power for use in thyroid carcinoma therapy.

Abstract Background Epigenetics has been known to play a critical role in regulating the malignant phenotype. This study was designed to examine the expression of DOT1L (histone 3 lysine 79 methyltransferase) and H3K79 methylation in normal ovarian tissues and ovarian tumors and to explore the function of DOT1L and its underline mechanisms in ovarian cancer. Methods The expression of DOT1L and H3K79 methylation in 250 ovarian tumor samples and 24 normal ovarian samples was assessed by immunohistochemistry. The effects of DOT1L on cell proliferation in vitro were evaluated using CCK8, colony formation and flow cytometry. The DOT1L-targeted genes were determined using chromatin immune-precipitation coupled with high-throughput sequencing (ChIP-seq) and ChIP-PCR. Gene expression levels were measured by real-time PCR and immunoblotting. The effects of DOT1L on tumor growth in vivo were evaluated using an orthotopic ovarian tumor model. Results DOT1L expression and H3K79 methylation was significantly increased in malignant ovarian tumors. High DOT1L expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, histologic grade, and lymphatic metastasis. DOT1L was an independent prognostic factor for the overall survival (OS) and progression-free survival (PFS) of ovarian cancer, and higher DOT1L expression was associated with poorer OS and PFS. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation; therefore, blocking DOT1L could result in G1 arrest and thereby impede the cell proliferation in vitro and tumor growth in vivo. Conclusions Our findings first demonstrate that DOT1L over-expression has important clinical significance in ovarian cancer and also clarify that it drives cell cycle progression through transcriptional regulation of CDK6 and CCND3 through H3K79 methylation, suggesting that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer.

Manipulating cell cycle is one of the common strategies used by viruses to generate favorable cellular environment to facilitate viral replication. Coxsackievirus B (CVB) is one of the major viral pathogens of human myocarditis and cardiomyopathy. Because of its small genome, CVB depends on cellular machineries for productive replication. However, how the structural and non-structural components of CVB would manipulate cell cycle is not clearly understood. In this study, we demonstrated that the capsid protein VP1 of CVB type 3 (CVB3) induced cell cycle arrest at G1 phase. G1 arrest was the result of the decrease level of cyclin E and the accumulation of p27Kip1. Study on the gene expression profile of the cells expressing VP1 showed that the expression of both heat shock protein 70-1 (Hsp70-1) and Hsp70-2 was significantly up-regulated. Knockdown of Hsp70 resulted in the increased level of cyclin E and the reduction of p27Kip1. We further demonstrated that the phosphorylation of the heat shock factor 1, which directly promotes the expression of Hsp70, was also increased in the cell expressing VP1. Moreover, we show that CVB3 infection also induced G1 arrest, likely due to dysregulating Hsp70, cyclin E, and p27, while knockdown of Hsp70 dramatically inhibited viral replication. Cell cycle arrest at G1 phase facilitated CVB3 infection, since viral replication in the cells synchronized at G1 phase dramatically increased. Taken together, this study demonstrates that the VP1 of CVB3 induces cell cycle arrest at G1 phase through up-regulating Hsp70. Our findings suggest that the capsid protein VP1 of CVB is capable of manipulating cellular activities during viral infection.

Niclosamide is a traditional anti-tapeworm drug that exhibits potent anti-cancer activity. Our previous study showed that niclosamide induces cell cycle arrest in G1 phase. Nevertheless, the underlying mechanism remains unknown. The following study investigated the molecular mechanism through which niclosamide induced G1 arrest in head and neck squamous cell carcinoma (HNSCC) cell lines. The effect of niclosamide on human HNSCC cell line WSU-HN6 and CNE-2Z were analyzed using IncuCyte ZOOMTM assay, flow cytometry (FCM), real-time PCR and western blot. Luciferase assay was conducted to demonstrate the interaction between let-7d (a let-7 family member which functions as a tumor suppressor by regulating cell cycle) and 3′UTR of CDC34 mRNA. Xenografts tumor model was established to evaluate the niclosamide treatment efficacy in vivo. Briefly, an exposure to niclosamide treatment led to an increased let-7d expression and a decreased expression of cell cycle regulator CDC34, finally leading to G1 phase arrest. Moreover, an overexpression of let-7d induced G1 phase arrest and downregulated CDC34, while the knockdown of let-7d partially rescued the niclosamide-induced G1 phase arrest. Luciferase assay confirmed the direct inhibition of CDC34 through the targeting of let-7d. Furthermore, niclosamide markedly inhibited the xenografts growth through up-regulation of let-7d and down-regulation of CDC34. To sum up, our findings suggest that niclosamide induces cell cycle arrest in G1 phase in HNSCC through let-7d/CDC34 axis, which enriches the anti-cancer mechanism of niclosamide.

A MEK1/2 inhibitor, binimetinib is promising as a therapeutic agent for malignant melanoma with N-RAS mutation. We examined in vitro effects of binimetinib on 10 human myeloid/lymphoid leukemia cell lines, and found that three of five cell lines with N-RAS mutation and one of five without N-RAS mutation were responsive to treatment with binimetinib. Binimetinib inhibited cell growth mainly by inducing G1 arrest and this action mechanism was assisted by gene set enrichment analysis. To identify signaling pathways associated with binimetinib response, we examined the status of MAP kinase/ERK and PI3Kinase/Akt pathways. The basal levels of phosphorylated ERK and Akt varied between the cell lines, and the amounts of phosphorylated ERK and Akt appeared to be reciprocal of each other. Interestingly, most of the binimetinib-resistant cell lines revealed strong Akt phosphorylation compared with binimetinib-sensitive ones. The effect of binimetinib may not be predicted by the presence/absence of N-RAS mutation, but rather by Akt phosphorylation status. Moreover, combination of binimetinib with a PI3K/Akt inhibitor showed additive growth-suppressive effects. These results suggest that binimetinib shows potential anti-leukemic effects and the basal level of phosphorylated Akt might serve as a biomarker predictive of therapeutic effect. [ABSTRACT FROM AUTHOR]

Manipulating cell cycle is one of the common strategies used by viruses to generate favorable cellular environment to facilitate viral replication. Coxsackievirus B (CVB) is one of the major viral pathogens of human myocarditis and cardiomyopathy. Because of its small genome, CVB depends on cellular machineries for productive replication. However, how the structural and non-structural components of CVB would manipulate cell cycle is not clearly understood. In this study, we demonstrated that the capsid protein VP1 of CVB type 3 (CVB3) induced cell cycle arrest at G1 phase. G1 arrest was the result of the decrease level of cyclin E and the accumulation of p27Kip1. Study on the gene expression profile of the cells expressing VP1 showed that the expression of both heat shock protein 70-1 (Hsp70-1) and Hsp70-2 was significantly up-regulated. Knockdown of Hsp70 resulted in the increased level of cyclin E and the reduction of p27Kip1. We further demonstrated that the phosphorylation of the heat shock factor 1, which directly promotes the expression of Hsp70, was also increased in the cell expressing VP1. Moreover, we show that CVB3 infection also induced G1 arrest, likely due to dysregulating Hsp70, cyclin E, and p27, while knockdown of Hsp70 dramatically inhibited viral replication. Cell cycle arrest at G1 phase facilitated CVB3 infection, since viral replication in the cells synchronized at G1 phase dramatically increased. Taken together, this study demonstrates that the VP1 of CVB3 induces cell cycle arrest at G1 phase through up-regulating Hsp70. Our findings suggest that the capsid protein VP1 of CVB is capable of manipulating cellular activities during viral infection. [ABSTRACT FROM AUTHOR]

The principal subtype of lung cancer, non-small cell lung cancer (NSCLC) is a life-threatening malignancy that causes high mortality rates. Bergapten (5-methoxypsoralen) has been identified to possess anticancer activity against a number of carcinomas. In the present study, the effects of bergapten on NSCLC cells were investigated. The cell viability was determined by MTT assay. Cell cycle distribution was analyzed using flow cytometry. Protein expression and kinase cascade were demonstrated using western blot analysis. The results demonstrated that treatment with bergapten (50 µM for 48 h) inhibited the viability of A549 and NCI-H460 NSCLC cells to 79.1±2.8% and 74.5±3.1%, respectively, compared with the controls. It was identified that bergapten induced G1 phase accumulation in A549 and NCI-H460 cells between ~58 and 75% (P<0.01). In addition, bergapten significantly increased the sub-G1 phase ratio to ~9% (P<0.05) in the two cell types. Further investigation demonstrated that bergapten upregulated the expression of cellular tumor antigen p53 (p53) and its downstream proteins cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor 1B, whereas, it downregulated the expression of cyclin D1 and CDK4. Overall, these results suggested that bergapten may inhibit cell viability and trigger G1 arrest and apoptosis in A549 and NCI-H460 cells, which may be attributed to the activation of p53-mediated cascades. Therefore, bergapten may be beneficial for NSCLC treatment. [ABSTRACT FROM AUTHOR]

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a cold-sensitive mutation, rpc128-1007, in the yeast gene encoding the second largest Pol III subunit, Rpc128. rpc128-1007 is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that rpc128-1007 mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most rpc128-1007 mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the rpc128-1007 mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double rpc128-1007 maf1Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with rpc128-1007 is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase. [ABSTRACT FROM AUTHOR]

Lipin-1 is an Mg2+-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1.

Multiple myeloma (MM) still remains an incurable disease, therefore discovery of novel drugs boosts the therapeutics for MM. The natural compound 1,2,3,4,6-Penta-O-galloyl-beta-D-glucopyranoside (PGG) has been shown to exhibit antitumor activities against various cancer cells. Here, we aim to evaluate antitumor effects of PGG on MM cell lines. PGG inhibited the growth of three different MM cell lines in a dose- and time-dependent manner. Cell cycle analysis revealed that PGG treatment caused cell cycle arrest in G1 phase. It also induced apoptosis which was indicated by significant increases of Annexin V positive cells, caspase 3/7 activity, and cleaved caspase 3 expression in PGG treated MM cell. Since MYC is frequently hyperactivated in MM and inhibition of MYC leads to MM cell death. We further demonstrated that PGG decreased MYC expression in protein and mRNA levels and reversed the mRNA expression of MYC target genes such as p21, p27, and cyclin D2. In addition, PGG also reduced protein expression of DEPTOR which is commonly overexpressed in MM. Unexpectedly, PGG antagonized the cytotoxic effect of bortezomib in the combination treatment. However, PGG treatment sensitized MM cells to another proteasome inhibitor MG132 induced cytotoxicity. Moreover, MYC inhibitor JQ1 enhanced the cytotoxic effect of bortezomib on MM cells. Our findings raised concerns about the combinatory use of bortezomib with particular types of chemicals. The evidence also provide useful insights into the combination of MYC and proteasome-inhibitors for MM therapy. Finally, PGG has a therapeutic potential for treatment of MM and further development is mandatory.

Ophiobolin O is a member of ophiobolin family, which has been proved to be a potent anti-tumor drug candidate for human breast cancer. However, the anti-tumor effect and the mechanism of ophiobolin O remain unclear. In this study, we further verified ophiobolin O-induced G1 phase arrest in human breast cancer MCF-7 cells, and found that ophiobolin O reduced the phosphorylation level of AKT and GSK3β, and induced down-regulation of cyclin D1. The inverse docking (INVDOCK) analysis indicated that ophiobolin O could bind to GSK3β, and GSK3β knockdown abolished cyclin D1 degradation and G1 phase arrest. Pre-treatment with phosphatase inhibitor sodium or thovanadate halted dephosphorylation of AKT and GSK3β, and blocked ophiobolin O-induced G1 phase arrest. These data suggest that ophiobolin O may induce G1 arrest in MCF-7 cells through interaction with AKT/GSK3β/cyclin D1 signaling. In vivo, ophiobolin O suppressed tumor growth and showed little toxicity in mouse xenograft models. Overall, these findings provide theoretical basis for the therapeutic use of ophiobolin O.

Current treatment strategies for leukemia still have some limitations such as severe side effects and drug resistance. Less toxic and more effective drugs for leukemia patients are therefore expected. In the present study, the efficacy of a dual PI3K/mTOR inhibitor, Voxtalisib, on acute myeloid leukemia (AML) cell line HL60 and chronic myeloid leukemia (CML) cell line K562, as well as their Adriamycin (ADR)-selected multi drug resistance (MDR) counterparts HL60/ADR and K562/A02, was investigated. Voxtalisib exhibited potent anti-proliferative activity on these four cell lines dose-dependently, with IC 50 values as 2.23 μM for HL60, 4.79 μM for HL60/ADR, 4.20 μM for K562 and 3.90 μM for K562/A02 cells. Voxtalisib arrested cell cycle progression at G1 phase in all cell lines by upregulating p27, downregulating cyclin D1 and p-pRb. When combined with ADR, Voxtalisib reversed the ADR-resistance of HL60/ADR and K562/A02 cells, possibly by reducing MDR1 and MRP1 expression. In conclusion, Voxtalisib showed anti-leukemia activity on AML and CML cell lines as well as their multidrug resistant ones, suggesting Voxtalisib might become a promising drug candidate for therapy of AML and CML in the future. [ABSTRACT FROM AUTHOR]

Abstract: Dinoflagellates of the genus Symbiodinium live in symbiosis with many invertebrates, including reef‐building corals. Hosts maintain this symbiosis through continuous regulation of Symbiodinium cell density via expulsion and degradation (postmitotic) and/or constraining cell growth and division through manipulation of the symbiont cell cycle (premitotic). Importance of premitotic regulation is unknown since little data exists on cell cycles for the immense genetic diversity of Symbiodinium. We therefore examined cell cycle progression for several distinct SymbiodiniumITS2‐types (B1, C1, D1a). All types exhibited typical microalgal cell cycle progression, G1 phase through to S phase during the light period, and S phase to G2/M phase during the dark period. However, the proportion of cells in these phases differed between strains and reflected differences in growth rates. Undivided larger cells with 3n DNA content were observed especially in type D1a, which exhibited a distinct cell cycle pattern. We further compared cell cycle patterns under different growth light intensities and thermal regimes. Whilst light intensity did not affect cell cycle patterns, heat stress inhibited cell cycle progression and arrested all strains in G1 phase. We discuss the importance of understanding Symbiodinium functional diversity and how our findings apply to clarify stability of host‐Symbiodinium symbioses. [ABSTRACT FROM AUTHOR]

Simvastatin (SIM), a widely used cholesterol‐lowering drug, also exhibits tumor‐suppressive potentials in several types of malignancy. Colorectal cancer (CRC), the third most common malignant neoplasm, accounts for the second most leading cause of cancer‐related deaths worldwide. In the present study, we investigated the anticancer effects of SIM on CRC using primary cancer cells lines (CPs: CP1 to CP5) isolated from five Taiwanese colorectal cancer patients as a model for colorectal cancer. We treated all five CPs with SIM for 24–72 hr and observed the respective cell viability by an MTT assay. SIM increased DNA content of the G1 phase, but did not induce apoptosis/necrosis in CPs as shown by flow cytometry with propidium iodide (PI)/annexin V double staining and PI staining. The expression of G1 phase‐related proteins was analyzed by RT‐PCR and Western blotting. SIM suppressed cell growth and induced cell cycle G1‐arrest by suppressing the expression of CDK4/cyclin D1 and CDK2/cyclin E1, but elevating the expression of glycogen synthase kinase 3β in CPs. Our findings indicate that SIM may have antitumor activity in established colorectal cancer. [ABSTRACT FROM AUTHOR]

Abstract: Cyclin‐dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin‐dependent kinase (CDK)‐cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654‐bp‐long BmCKI‐L (the longer splice variant) encoding a protein with 217 amino acids and a 579‐bp‐long BmCKI‐S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI‐L and BmCKI‐S contain the Cip/Kip family conserved cyclin‐binding domain and the CDK‐binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181–210. Overexpression of BmCKI‐L or BmCKI‐S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI‐L or BmCKI‐S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI‐L and BmCKI‐S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI‐L overexpression (BmCKI‐L‐OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI‐L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) still remains an incurable disease, therefore discovery of novel drugs boosts the therapeutics for MM. The natural compound 1,2,3,4,6-Penta-Ogalloyl- beta-D-glucopyranoside (PGG) has been shown to exhibit antitumor activities against various cancer cells. Here, we aim to evaluate antitumor effects of PGG on MM cell lines. PGG inhibited the growth of three different MM cell lines in a dose- and time-dependent manner. Cell cycle analysis revealed that PGG treatment caused cell cycle arrest in G1 phase. It also induced apoptosis which was indicated by significant increases of Annexin V positive cells, caspase 3/7 activity, and cleaved caspase 3 expression in PGG treated MM cell. Since MYC is frequently hyperactivated in MM and inhibition of MYC leads to MM cell death. We further demonstrated that PGG decreased MYC expression in protein and mRNA levels and reversed the mRNA expression of MYC target genes such as p21, p27, and cyclin D2. In addition, PGG also reduced protein expression of DEPTOR which is commonly overexpressed in MM. Unexpectedly, PGG antagonized the cytotoxic effect of bortezomib in the combination treatment. However, PGG treatment sensitized MM cells to another proteasome inhibitor MG132 induced cytotoxicity. Moreover, MYC inhibitor JQ1 enhanced the cytotoxic effect of bortezomib on MM cells. Our findings raised concerns about the combinatory use of bortezomib with particular types of chemicals. The evidence also provide useful insights into the combination of MYC and proteasome-inhibitors for MM therapy. Finally, PGG has a therapeutic potential for treatment of MM and further development is mandatory. [ABSTRACT FROM AUTHOR]

The checkpoint with forkhead‑associated (FHA) domain and RING‑finger (CHFR) protein was identified as a cell cycle checkpoint protein and E3 ubiquitin ligase. In the present study, the potential functions of CHFR in pancreatic cancer were investigated. CHFR expression was measured in five pancreatic cancer cell lines by reverse transcriptionquantitative polymerase chain reaction and western blotting. Capan‑1 cells stably expressing CHFR were established by lentiviral vector transfection. Cell proliferation was assessed using Cell Counting Kit‑8, and cell migration/invasion assay was determined using Transwell assays. Cell cycle and apoptosis induced by gemcitabine or docetaxel were evaluated using flow cytometry. CHFR expression levels were also evaluated in pancreatic ductal adenocarcinoma (PDAC) tumor samples as well as adjacent non‑tumor tissues by immunohistochemistry. The significance of CHFR expression was determined, with respect to clinicopathological features and overall survival. Overexpression of CHFR in Capan‑1 cells led to a decreased proliferative rate and reduced cell migration and invasion abilities. Results also indicated an increase in G1 phase cells in Capan‑1 cells overexpressing CHFR. Docetaxel‑induced apoptosis was inhibited in Capan‑1 cells with CHFR‑overexpression. A reduction in CHFR expression was detected in 51.9% of patients with PDAC, which significantly correlated with later T‑stage. The results show CHFR functions as a tumor suppressor in pancreatic cancer, suggests its potential role in controlling the cell cycle of pancreatic cancer cells; however, CHFR overexpression is not a favorable factor in apoptosis induced by docetaxel. [ABSTRACT FROM AUTHOR]

Background/Aims: CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types; however, its prognostic impact and role in cellular processes, particularly in thyroid carcinoma, are still unclear. Methods: To elucidate the prognostic impact of CRIP1, we analyzed tissues from 58 primary invasive thyroid carcinomas using immunohistochemistry. Western blotting was performed to investigate CRIP1 protein expression in the thyrocyte cell line Nthyori 3-1 and four different thyroid carcinoma cell lines, K1, TPC-1, TT, and SW579. Endogenous expression of CRIP1 was suppressed using a siRNA (si-CRIP1). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate cell viability. Flow cytometric analysis was used to detect cell cycle progression and cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion were detected using the transwell assay. Results: The immunohistochemistry results showed that CRIP1 was overexpressed in thyroid carcinoma. CRIP1 expression was associated with tumor size, TNM stage, and lymphatic metastasis, but not with age, gender, and tumor location. In addition, the expression of CRIP1 in K1, TPC-1, TT, and SW529 cells was higher than that in the Nthy-ori 3-1 cells. The highest expression was observed in the SW579 and TT cells. Furthermore, silencing CRIP1 inhibited the proliferation, migration, and invasion of thyroid carcinoma cell lines SW579 and TT. We also found that silencing CRIP1 induced G1 arrest and apoptosis of thyroid carcinoma cell lines SW579 and TT. Conclusion: In conclusion, CRIP1 acts as an oncogene in the cell proliferation, migration, and invasion processes of thyroid carcinoma. CRIP1 may serve well as an independent prognostic marker with significant predictive power for use in thyroid carcinoma therapy. [ABSTRACT FROM AUTHOR]

Although fucoidan has been shown to exert anticancer activity against several types of cancer cell lines, no reports have explored fucoidan-affected cell growth in human urinary bladder cancer cells. In this study, we investigated the anti-proliferative effects of fucoidan in human bladder cancer T24 cells. Our results indicated that fucoidan decreased the viability of T24 cells through the induction of G1 arrest and apoptosis. Fucoidan-induced G1 arrest is associated with the enhanced expression of the Cdk inhibitor p21WAF1/CIP1 and dephosphorylation of the pRB along with enhanced binding of p21 to Cdk4/6 as well as pRB to the transcription factor E2Fs. Further investigations showed the loss of mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol, proving mitochondrial dysfunction upon fucoidan treatment with a corresponding increase in the Bax/Bcl-2 expression ratio. Fucoidan-triggered apoptosis was also accompanied by the up-regulation of Fas and truncated Bid as well as the sequential activation of caspase-8. Furthermore, a significant increased activation of caspase-9/-3 was detected in response to fucoidan treatment with the decreased expression of IAPs and degradation of PARP, whereas a pan-caspase inhibitor significantly suppressed apoptosis and rescued the cell viability reduction. In conclusion, these observations suggest that fucoidan attenuates G1-S phase cell cycle progression and serves as an important mediator of crosstalk between caspase-dependent intrinsic and extrinsic apoptotic pathways in T24 cells.