Phase Cell Cycle Progression Research Articles

FBXW8 regulates G1 and S phases of cell cycle progression by restricting β-TrCP1 function.

Sequential alteration in the expression levels of cell cycle regulatory proteins is crucial for faithful cell cycle progression to maintain the cellular homeostasis. F-box protein β-TrCP1 is known to control the expression levels of several important cell cycle regulatory proteins. However, how the function of β-TrCP1 is regulated in spatiotemporal manner during cell cycle progression remains elusive. Here, we show that expression levels of β-TrCP1 oscillate during cell cycle progression with a minimum level at the G1 and S phases of cell cycle. Using biochemical, flow cytometry, and immunofluorescence techniques, we found that oscillation of β-TrCP1 expression is controlled by another F-box protein FBXW8. FBXW8 directs the proteasomal degradation of β-TrCP1 in MAPK pathway-dependent manner. Interestingly, we found that the attenuation of β-TrCP1 by FBXW8 is important for Cdc25A-mediated cell cycle transition from G1 phase to S phase as well as DNA damage-free progression of S phase. Overall, our study reveals the intriguing molecular mechanism and significance of maintenance of β-TrCP1 levels during cell cycle progression by FBXW8-mediated proteasomal degradation.

Kaempferol Reverses Aerobic Glycolysis via miR-339-5p-Mediated PKM Alternative Splicing in Colon Cancer Cells.

Colon cancer is an aggressive malignancy with very limited therapeutic approaches. The available therapeutic agents for colon cancer show strong adverse effects and poor effectiveness, indicating the urgent need to identify new therapeutic drugs for this malignancy. Kaempferol, a flavonoid found in a variety of natural foods, exhibits significant inhibitory effects on colon cancer. Here, it was found that kaempferol inhibited the proliferation of human colon cancer cells HCT116 and DLD1 in a dose-dependent manner, and the IC50 values were 63.0 ± 12.9 and 98.3 ± 15.9 μM, respectively. Also, kaempferol treatment delayed G1 phase progression of cell cycle and induced apoptosis. Aerobic glycolysis is the major energy source for various tumor growths, including colon cancer. Indeed, kaempferol treatment impaired glucose consumption, which subsequently led to reduced lactic acid accumulation and ATP production. Mechanistically, kaempferol promoted the expression of miR-339-5p. Further studies identified hnRNPA1 and PTBP1 as two direct targets of miR-339-5p. By directly targeting hnRNPA1 and PTBP1, miR-339-5p reduced the expression of M2-type pyruvate kinase (PKM2) but induced that of PKM1. In conclusion, these data demonstrate that by modulating miR-339-5p-hnRNPA1/PTBP1-PKM2 axis, kaempferol inhibits glycolysis and colon cancer growth, which reveals a new explanation for the molecular mechanism underlying kaempferol anti-tumor.

E2F1 Induces KIF26A Transcription and Promotes Cell Cycle Progression via CDK-RB-E2Fs Feedback Loop in Breast Cancer.

ObjectiveThe aim of this study was to investigate the role of KIF26A in breast cancer.MethodqRT-PCR and immunohistochemistry were conducted to explore KIF26A expression and functional contribution to breast cancer development. MTS, EDU, colony formation assays, and flow cytometry analysis were conducted to assess cell proliferation characteristics and cell cycle progression. A series of 5′-flanking region deletion plasmids and mutating the binding site, with the luciferase reporter assay, were used to identify the core promotor region of KIF26A. The prediction by software and construction of the transcriptional factor plasmids were used to identify the transcriptional factor. Chromatin immunoprecipitation assay could demonstrate transcriptional factor directly binding to the KIF26A promoter. Human Genome Oligo Microarray Assay and gene ontology (GO) and pathway analyses were used to predict the downstream pathway.ResultsOur results showed that in breast cancer tissues, elevated KIF26A expression was significantly correlated with lymph node metastasis. KIF26A could promote proliferation and G0/G1 phase cell cycle progression in breast cancer cells. The core promoter region of the human KIF26A gene was located upstream of the transcription start site at position −395 to −385. The transcriptional factor E2F1 was shown to activate KIF26A expression. Furthermore, KIF26A was shown to inhibit the expression of p21, then activate CDK–RB–E2Fs pathway. The elevated E2F1 can activate the cell cycle progression and the KIF26A expression, forming feedback loop.ConclusionsThe present study demonstrated that KIF26A, directly upregulated by E2F1, promoted cell proliferation and cell cycle progression via CDK–RB–E2Fs feedback loop in breast cancer.

Sustained high expression of NRF2 and its target genes induces dysregulation of cellular proliferation and apoptosis is associated with arsenite-induced malignant transformation of human bronchial epithelial cells

In arsenic toxicity, activation of the erythroid 2-related factor 2 (NRF2) pathway is regarded as a driver of cancer development and progression; however, the mechanisms by which NRF2 gene expression regulates cell cycle progression and mediates pathways of cellular proliferation and apoptosis in arsenic-induced lung carcinogenesis are poorly understood. In this study, we explored the regulatory functions of NRF2 expression and its target genes in immortalized human bronchial epithelial (HBE) cells continuously exposed to 1.0 μM sodium arsenite over approximately 43 passages (22 weeks). The experimental treatment induced malignant transformation in HBE cells, characterized by increased cellular proliferation and soft agar clone formation, as well as cell migration, and accelerated cell cycle progression from G0/G1 to S phase with increased levels of cyclin E-CDK2 complex,decreased cellular apoptosis rate. Moreover, we observed a sustained increase in NRF2 protein levels and those of its target gene products (NQO1, BCL-2) with concurrently decreased expression of apoptosis-related proteins (BAX, Cleaved-caspase-3/Caspase-3 and CHOP) and increased expression of the anti-apoptotic protein MCL-1. Silencing NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HBE) cells was shown to reverse the malignant phenotype. Further, siRNA silencing of NQO1 significantly decreased levels of the cyclin E-CDK2 complex, inhibiting G0/G1 to S phase cell cycle progression and transformation to the T-HBE phenotypes. This study demonstrated a novel role for the NRF2/NQO1 signaling pathway in mediating arsenite-induced cell transformation by increasing the expression of cyclin E-CDK2, and accelerating the cell cycle and cell proliferation. Arsenite promotes activation of the NRF2/BCL-2 signaling pathway inhibited CHOP increasing cellular resistance to apoptosis and further promoting malignant transformation.

Calreticulin increases growth and progression of natural killer/T-cell lymphoma.

In this study, we investigated the role of calreticulin (CALR) in the pathogenesis of natural killer/T-cell lymphoma (NKTCL). CALR expression was significantly higher in the NKTCL tissues than normal control tissues in the GSE80632 dataset. High CALR expression correlated with poorer overall survival of NKTCL patients (P = 0.0248). CALR mRNA and protein levels were significantly higher in NKTCL cell lines (NK92, SNK6, and SNT8) than normal NK cells. CALR-silenced SNK6 cells generated significantly smaller xenograft tumors in immunodeficient NCG mice than control SNK6 cells. CALR-knockdown NKTCL cells showed significantly less in vitro proliferation and Transwell migration than the controls. CALR knockdown inhibited G1-to-S phase cell cycle progression by increasing the levels of p27 cell cycle inhibitor and reducing the levels of cyclin E2 and cyclin-dependent kinase 2 (CDK2). CALR knockdown inhibited epithelial-to-mesenchymal transition (EMT) by decreasing the levels of β-catenin and TCF/ZEB1 and upregulating E-cadherin. These data demonstrate that CALR regulates the growth and progression of NKTCL cells by modulating G1-to-S cell cycle progression and EMT.

CTIM-10. PHASE II STUDY OF PEMBROLIZUMAB PLUS SurVaxM FOR GLIOBLASTOMA AT FIRST RECURRENCE

Abstract BACKGROUND Pembrolizumab is a potent humanized immunoglobulin G4 monoclonal antibody with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its interaction with programmed cell death ligand 2 (PD-L2). Survivin is a 16.5 kDa intracellular protein that belongs to the inhibitor of apoptosis protein (IAP) family. It acts in concert with the mitotic spindle apparatus to regulate cell division and localizes to the spindle microtubule organizing center (MTOC) during the G2/M phase of cell cycle progression. Survivin has also been shown to modulate the function of a number of terminal effector cell death proteases (caspases) leading to an inhibition of apoptosis. METHODS This is a Phase II study of two arms in patients with recurrent glioblastoma. Arm A is patients with first recurrence of glioblastoma who have failed prior chemotherapy and radiation but have not received any immunotherapy. Arm B is an exploratory arm of 10 patients who have failed prior anti-PD1 therapy. The ongoing study is a phase II clinical study with a 10 patient, toxicity run-in. All patients will receive the study drug combination consisting of SurVaxM and pembrolizumab with no randomization, stratification or dose escalation. RESULTS So far ten patients have been enrolled on the study as safety run in. Primary endpoint is Progression free survival at 6 months. Safety and tolerability of Pembrolizumab and SurVaxM, Response rates of Pembrolizumab and SurVaxM determined using RANO criteria are secondary endpoints. Additional secondary endpoints include Overall survival and Progression Free survival Exploratory endpoints include Cellular and humoral immune responses during concurrent administration of Pembrolizumab and SurVaxM. CONCLUSION This is an ongoing clinical trial.

Human P2X7 Receptor Causes Cycle Arrest in RPMI-8226 Myeloma Cells to Alter the Interaction with Osteoblasts and Osteoclasts.

Multiple myeloma is a malignant expansion of plasma cells and aggressively affects bone health. We show that P2X7 receptor altered myeloma growth, which affects primary bone cells in vitro. Expression on six human myeloma cell lines confirmed the heterogeneity associated with P2X7 receptor. Pharmacology with 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) as agonist showed dose-dependent membranal pores on RPMI-8226 (p = 0.0027) and blockade with P2X7 receptor antagonists. Ca2+ influx with increasing doses of BzATP (p = 0.0040) was also inhibited with antagonists. Chronic P2X7 receptor activation reduced RPMI-8226 viability (p = 0.0208). No apoptosis or RPMI-8226 death was observed by annexin V/propidium iodide (PI) labeling and caspase-3 cleavage, respectively. However, bromodeoxyuridine (BrdU) labelling showed an accumulation of RPMI-8226 in the S phase of cell cycle progression (61.5%, p = 0.0114) with significant decline in G0/G1 (5.2%, p = 0.0086) and G2/M (23.5%, p = 0.0015) phases. As myeloma pathology depends on a positive and proximal interaction with bone, we show that P2X7 receptor on RPMI-8226 inhibited the myeloma-induced suppression on mineralization (p = 0.0286) and reversed the excessive osteoclastic resorption. Our results demonstrate a view of how myeloma cell growth is halted by P2X7 receptor and the consequences on myeloma–osteoblast and myeloma–osteoclast interaction in vitro.

A comprehensive fluorescent sensor for spatiotemporal cell cycle analysis in Arabidopsis.

Assessing cell proliferation dynamics is crucial to understand the spatiotemporal control of organogenesis. Here we have generated a versatile fluorescent sensor, PlaCCI (plant cell cycle indicator) on the basis of the expression of CDT1a-CFP, H3.1-mCherry and CYCB1;1-YFP, that identifies cell cycle phases in Arabidopsis thaliana. This tool works in a variety of organs, and all markers and the antibiotic resistance are expressed from a single cassette, facilitating the selection in mutant backgrounds. We also show the robustness of PlaCCI line in live-imaging experiments to follow and quantify cell cycle phase progression.

The crosstalk between platelets and body fat: A reverse translational study

Our previous study found that platelet counts were positively associated with body fat percentage in human. In the present study, we conducted a reverse translational study to explore the role of platelets in modulating pre-adipocyte proliferation in mice. Mouse pre-adipocyte cell line (3T3-L1) and human pre-adipocytes harvested from female subcutaneous fat were used. Pre-adipocytes were co-cultured with platelets or platelet releasate, which were isolated from mice or humans. The cell viability and proliferative ability of the pre-adipocytes were examined by MTT and flow cytometry assays. Western blotting analysis was used to determine the phosphorylation levels of proteins in the mTOR pathway. The number of platelets in the adipose tissues from obese mice was significantly higher than that from lean mice. Platelets and collagen-activated platelet releasate stimulated the proliferation of human pre-adipocytes and 3T3-L1 cells invitro. Besides, platelets from obese mice were more potent in stimulating pre-adipocyte proliferation than those from lean control mice. Mechanistically, platelets enhanced pre-adipocyte proliferation through the acceleration of cell cycle progression from G0/G1 to S phase cell cycle progression. At the molecular level, platelets promoted pre-adipocyte proliferation through mTOR pathway-mediated upregulation of cyclin D1 expression. In conclusion, platelets and platelet releasate play an important role in the proliferation of pre-adipocytes. Our study may provide new clues and the molecular mechanism of the causal pathways between platelets and body fat to explain the finding we observed in population study.

Modulatory Effect of Indoles on the Expression of miRNAs Regulating G1/S Cell Cycle Phase in Breast Cancer Cells.

Indole-3-carbinol (I3C) is a naturally occurring glucosinolate found in Brassica vegetables that is usually converted in gastric acidic environment to the efficient metabolite 3,3'-diindolylmethane (DIM). Both indoles (I3C and DIM) are known chemopreventive agents for various cancers including breast cancer. This study aimed to investigate the influence of both indoles on the tumor suppressor miRNAs (let-7a-e, miR-15a, miR-16, miR-17-5p, miR-19a, and miR-20a) and oncomiRs (miR-181a, miR-181b, miR-210, miR-221, and miR-106a), which are controlling the cell cycle key regulators: cyclin-dependent kinases (CDKs), CDK inhibitor p27Kip1, and cyclin D1. Our results indicated that both indoles generally elevated the expression of the tumor suppressor miRNAs let-7a-e, miR-19a, miR-17-5p, and miR-20a and decreased the expression of the oncomiR list. Both indoles were able to significantly suppress the expression of CDK4 and CDK6 as well as the apoptotic markers Bcl-2 and survivin. Both indoles decreased cyclin-D1 protein, where I3C decreased cytoplasmic and nuclear cyclin-D1 significantly. Cytoplasmic and nuclear P27Kip1 showed overexpression following treatment with I3C higher than that detected following DIM treatment. This study provides a mechanistic elucidation of the previously reported cell cycle arrest by I3C and DIM in breast cancer cells suggesting that this effect could be through modulation of miRNAs expression that, in turn, regulates the genetic network controlling the G1/S phase in cell cycle progression.

Ni(II) curcumin complexes for cellular imaging and photo-triggered in vitro anticancer activity

Nickel(II) complexes [Ni(cur)(L)2](OAc) (1-3) where L is N,N-donor heterocyclic bases namely 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq in 2), dipyrido[3,2-a:2′,3′-c]phenazine (dppz in 3) and Hcur is curcumin were prepared, fully characterized and light-induced in vitro anticancer activity studied. Three nickel(II) complexes containing acetylacetonato (Hacac) ligand, viz.[Ni(acac)(L)2](OAc) (4-6) where L is phen (in 4), dpq (in 5), dppz (in 6) were prepared and used as controls. Complex 4 was structurally characterized by single crystal X-ray diffraction technique, which revealed an octahedral NiN4O2 geometry around the metal centre. Complexes 1-3 showed an intense curcumin-based band at ∼440 nm in DMSO-Tris-HCl buffer (pH = 7.2) (1:4 v/v) which masks the nickel based d-d band. The curcumin comlexes (1-3) were redox inactive at the nickel centre, whereas the acetylacetonato complexes (4-6) displayed an irreversible voltammetric response at ∼1.00 V vs. Ag/AgCl reference electrode in DMF. The complexes bind to calf thymus DNA (ct-DNA) with considerable affinity and interacted with human serum albumin (HSA) with moderate affinity. The Ni(II) curcumin complexes display significant in vitro light-induced cytotoxicity in HeLa (human cervical carcinoma) and A549 (lung cancer cells) involving reactive oxygen species (ROS), with very low dark toxicity. The complexes were found to be much less toxic to immortalized lung epithelial normal cells (HPL1D). Confocal microscopic images using complex 2 and 3 showed that they primarily localize in the cytosol of A549 cells. The mechanism of cell death is mainly apoptosis in nature showing arrest of sub-G1 phase of cell cycle progression in A549 cells under visible light exposure and involves significant loss of mitochondrial membrane potential as observed from JC-1 assay.

The (+)-Brevipolide H Displays Anticancer Activity against Human Castration-Resistant Prostate Cancer: The Role of Oxidative Stress and Akt/mTOR/p70S6K-Dependent Pathways in G1 Checkpoint Arrest and Apoptosis.

Because conventional chemotherapy is not sufficiently effective against prostate cancer, various examinations have been performed to identify anticancer activity of naturally occurring components and their mechanisms of action. The (+)-brevipolide H, an α-pyrone-based natural compound, induced potent and long-term anticancer effects in human castration-resistant prostate cancer (CRPC) PC-3 cells. Flow cytofluorometric analysis with propidium iodide staining showed (+)-brevipolide H-induced G1 arrest of cell cycle and subsequent apoptosis through induction of caspase cascades. Since Akt/mTOR pathway has been well substantiated in participating in cell cycle progression in G1 phase, its signaling and downstream regulators were examined. Consequently, (+)-brevipolide H inhibited the signaling pathway of Akt/mTOR/p70S6K. The c-Myc inhibition and downregulation of G1 phase cyclins were also attributed to (+)-brevipolide H action. Overexpression of myristoylated Akt significantly rescued mTOR/p70S6K and downstream signaling under (+)-brevipolide H treatment. ROS and Ca2+, two key mediators in regulating intracellular signaling, were determined, showing that (+)-brevipolide H interactively induced ROS production and an increase of intracellular Ca2+ levels. The (+)-Brevipolide H also induced the downregulation of anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL) and loss of mitochondrial membrane potential, indicating the contribution of mitochondrial dysfunction to apoptosis. In conclusion, the data suggest that (+)-brevipolide H displays anticancer activity through crosstalk between ROS production and intracellular Ca2+ mobilization. In addition, suppression of Akt/mTOR/p70S6K pathway associated with downregulation of G1 phase cyclins contributes to (+)-brevipolide H-mediated anticancer activity, which ultimately causes mitochondrial dysfunction and cell apoptosis. The data also support the biological significance and, possibly, clinically important development of natural product-based anticancer approaches.

Inhibition of PI3K/AKT molecular pathway mediated by membrane estrogen receptor GPER accounts for cryptotanshinone induced antiproliferative effect on breast cancer SKBR-3 cells

BackgroundBreast cancer is the most frequently diagnosed malignancy among women and the second leading cause of cancer death worldwide. Among which nuclear estrogen receptor (nER) negative breast cancer is always with much poor prognosis. Recently, membrane G protein coupled estrogen receptor (GPER), a newly recognized estrogen receptor has been documented to take essential part in the development and treatment of breast cancer. The present study was designed to investigate the anti nER negative breast cancer effect of cryptotanshinone (CPT), an important active compound of traditional Chinese medicine Danshen and its possible molecular pathway.MethodsThe following in vitro tests were performed in nER negative but GPER positive breast cancer SKBR-3 cells. The effect of CPT on cell proliferation rate and cell cycle distribution was evaluated by MTT cell viability test and flow cytometry assay respectively. The role of PI3K/AKT pathway and the mediated function of GPER were tested by western blot and immunofluorescence. Technique of gene silence and the specific GPER agonist G-1 and antagonist G-15 were employed in the experiments to further verify the function of GPER in mediating the anticancer role of CPT.ResultsThe results showed that proliferation of SKBR-3 cells could be blocked by CPT in a time and dose dependent manner. CPT could also exert antiproliferative activities by arresting cell cycle progression in G1 phase and down regulating the expression level of cyclin A, cyclin B, cyclin D and cyclin-dependent kinase 2 (CDK2). The antiproliferative effect of CPT was further enhanced by G-1 and attenuated by G-15. Results of western blot and immunofluorescence showed that expression of PI3K and p-AKT could be downregulated by CPT and such effects were mediated by GPER which were further demonstrated by gene silence test.ConclusionThe current study showed that the antiproliferative action of CPT on SKBR-3 cells was realized by inhibition of GPER mediated PI3K/AKT pathway. These findings provide further validation of GPER serving as useful therapeutic target.

Type 5 17-hydroxysteroid dehydrogenase/prostaglandin F synthase (AKR1C3) inhibition and potential anti-proliferative activity of cholest-4-ene-3,6-dione in MCF-7 breast cancer cells

Cholest-4-ene-3,6-dione (KS) is a cholesterol oxidation product which exhibits anti-proliferative activity. However, its precise mechanism of action remains unknown. In this study, the effects of KS on AKR1C3 inhibition and anti-proliferative activities were investigated in the hormone-dependent MCF-7 breast cancer cells. We identified that KS arrested the enzymatic conversion of estrone to 17-β estradiol, by inhibiting AKR1C3 in intact MCF-7 cells. The anti-proliferative effects of KS were evaluated by MTT assay, acridine orange and ethidium bromide dual staining, cell cycle analysis and Western blotting. KS arrested the cell cycle progression in the G1 phase with a concomitant increase of the Sub-G0 population to increase in concentration and time. It also enhanced the p53 and NFkB expression and induced caspase-12, 9 and 3 processing and down-regulated the Bcl-2 expression. Molecular docking studies performed to understand the inhibition mechanism of KS on AKR1C3 revealed that KS occupied the binding region of AKR1C3 with almost similar orientation as indomethacin (IM), thereby acting as an antagonistic agent for AKR1C3. Based on the results it is identified that KS induces inhibition of AKR1C3 and cell death in MCF-7 cells. These results indicate that KS can be used as a molecular scaffold for further development of novel small-molecules with better specificity towards AKR1C3.

Downregulated LINC00628 aggravates the progression of colorectal cancer via inhibiting p57 level.

To uncover the potential function of LINC00628 in influencing the progression of colorectal cancer (CRC) and its underlying mechanism. Relative levels of LINC00628 and p57 in CRC tissues and cell lines were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Regulatory effects of LINC00628 and p57 on proliferation, cell cycle, and apoptosis of SW480 and SW620 cells were assessed. Subcellular distribution of LINC00628 in CRC cells was analyzed. Moreover, the interaction between LINC00628 and enhancer of zeste homolog 2 (EZH2) was evaluated by the RNA Binding Protein Immunoprecipitation (RIP) assay. LINC00628 was downregulated in CRC tissues and cell lines. CRC patients expressing a low level of LINC00628 suffered worse prognosis. The. knockdown of LINC00628 enhanced proliferative ability, prolonged S phase in cell cycle progression, and inhibited apoptosis in SW480 and SW620 cells. LINC00628 was mainly distributed in the nucleus. The RIP assay demonstrated that LINC00628 could bind to EZH2 to upregulate the p57 level. Rescue experiments verified that the overexpression of p57 could reverse regulatory effects of downregulated LINC00628 on proliferative and apoptotic abilities of CRC. LINC00628 is downregulated in CRC. It aggravates the progression of CRC by binding to EZH2 to further inhibit the p57 level.

Ending Restenosis: Inhibition of Vascular Smooth Muscle Cell Proliferation by cAMP.

Increased vascular smooth muscle cell (VSMC) proliferation contributes towards restenosis after angioplasty, vein graft intimal thickening and atherogenesis. The second messenger 3′ 5′ cyclic adenosine monophosphate (cAMP) plays an important role in maintaining VSMC quiescence in healthy vessels and repressing VSMC proliferation during resolution of vascular injury. Although the anti-mitogenic properties of cAMP in VSMC have been recognised for many years, it is only recently that we gained a detailed understanding of the underlying signalling mechanisms. Stimuli that elevate cAMP in VSMC inhibit G1-S phase cell cycle progression by inhibiting expression of cyclins and preventing S-Phase Kinase Associated Protein-2 (Skp2-mediated degradation of cyclin-dependent kinase inhibitors. Early studies implicated inhibition of MAPK signalling, although this does not fully explain the anti-mitogenic effects of cAMP. The cAMP effectors, Protein Kinase A (PKA) and Exchange Protein Activated by cAMP (EPAC) act together to inhibit VSMC proliferation by inducing Cyclic-AMP Response Element Binding protein (CREB) activity and inhibiting members of the RhoGTPases, which results in remodelling of the actin cytoskeleton. Cyclic-AMP induced actin remodelling controls proliferation by modulating the activity of Serum Response Factor (SRF) and TEA Domain Transcription Factors (TEAD), which regulate expression of genes required for proliferation. Here we review recent research characterising these mechanisms, highlighting novel drug targets that may allow the anti-mitogenic properties of cAMP to be harnessed therapeutically to limit restenosis.

MiRNA-616 aggravates the progression of bladder cancer by regulating cell proliferation, migration and apoptosis through downregulating SOX7.

To investigate the regulatory effect of microRNA-616 (miRNA-616) on cellular behaviors of bladder cancer and the potential mechanism. The expressions of miRNA-616 and SOX7 in bladder cancer tissues and cell lines were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between miRNA-616 and SOX7 was assessed through Dual-Luciferase Reporter Gene Assay. The regulatory effects of miRNA-616 and SOX7 on cellular behaviors of bladder cancer were evaluated through cell counting kit-8 (CCK-8), colony formation, transwell migration assay, and flow cytometry. MiRNA-616 was upregulated, whereas SOX7 was downregulated in bladder cancer tissues and cell lines. The silence of miRNA-616 attenuated the proliferative and migratory abilities, arrested cell cycle progression in the G2 phase, and stimulated apoptosis in UMUC3 and T24 cells. SOX7 was the target gene of miRNA-616, and its level was negatively regulated by miRNA-616. The knockdown of SOX7 enhanced the proliferative and migratory abilities, and attenuated apoptosis of bladder cancer cells. MiRNA-616 accelerates bladder cancer cells to proliferate and migrate and inhibits apoptosis by downregulating SOX7. MiRNA-616/SOX7 may be potential therapeutic targets for bladder cancer.

Co-targeting PI3K/Akt and MAPK/ERK pathways leads to an enhanced antitumor effect on human hypopharyngeal squamous cell carcinoma.

The present study aims to determine whether co-targeting PI3K/Akt and MAPK/ERK pathways in human hypopharyngeal squamous cell carcinoma (HSCC) is a potential anticancer strategy. We retrospectively analyzed the clinical data of HSCC patients, and the phosphorylation status of Akt and Erk in HSCC and tumor adjacent tissues was evaluated by immunohistochemistry. MTT and colony formation assay were performed to determine the anti-proliferative effect of PI3K/mTOR inhibitor GDC-0980 and MEK inhibitor Refametinib on HSCC cell line Fadu. Wound-healing and Transwell migration assay were used to analyze the anti-migrative capability of the two drugs. The involved anti-tumor mechanism was explored by flow cytometry, qRT-PCR and western blot. The combinational anticancer effect of GDC-0980 and Refametinib was evaluated according to Chou and Talalay's method. The levels of p-Akt and p-Erk were increased significantly with the progression of clinical stage of HSCC, suggesting PI3K/Akt and MAPK/ERK pathways might be associated with HSCC occurrence and progression. Furthermore, both GDC-0980 and Refametinib showed obvious antitumor effects on FaDu cells. Treatment by the two drugs arrested FaDu cell cycle progression in G1 phase, with reduction of cyclin D1 and p-Rb, in contrast to enhancement of p27. GDC-0980 inhibited FaDu cell migration and reduced metastasis related proteins including p-PKCζ, p-Integrin β1 and uPA. Combination use of GDC-0980 and Refametinib exhibited strong synergistic anti-tumor effect. Dual inhibition of PI3K/Akt and MAPK/ERK pathway by GDC-0980 and Refametinib might be a promising treatmentstrategy for HSCC patients.

Silencing Lysine-Specific Histone Demethylase 1 (LSD1) Causes Increased HP1-Positive Chromatin, Stimulation of DNA Repair Processes, and Dysregulation of Proliferation by Chk1 Phosphorylation in Human Endothelial Cells.

The methylation of histone lysine residues modifies chromatin conformation and regulates the expression of genes implicated in cell metabolism. Lysine-specific demethylase 1 (LSD1) is a flavin-dependent monoamine oxidase that can demethylate mono- and dimethylated histone lysines 4 and 9 (H3K4 and H3K9). The removal of methyl groups from the lysine residues of histone and non-histone proteins was found to be an important regulatory factor of cell proliferation. However, its role has not been fully elucidated. In this study, we assessed LSD1-mediated cell cycle progression using a human endothelial cell model. The short hairpin RNA knockdown of LSD1 inhibits the G2/M phase of cell cycle progression by checkpoint kinase 1 (Chk1) phosphorylation (S137). We observed elevated DNA damage, which was consistent with the increased detection of double-strand breaks as well as purines and pyrimidines oxidation, which accompanied the activation of ATR/ATRIP signaling by H2AXS139 phosphorylation. The irreversible pharmacological inhibition of LSD1 by 2-phenylcyclopropylamine (2-PCPA) inactivated its enzymatic activity, causing significant changes in heterochromatin and euchromatin conformation assessed by chromatin assembly factor 1 subunit A (CAF1A) and heterochromatin protein 1 isoform α and γ (HP1α/γ) immunofluorescence analysis. We conclude that the knockdown of LSD1 in endothelial cells leads to increased HP1-positive chromatin, the stimulation of DNA repair processes, and the dysregulation of proliferation machinery.

Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells.

Reactive oxygen species (ROS) which lead to oxidative stress affect ovarian function. Grape seed extract (GSE) could be proposed as an effective antioxidant, particularly due to its proanthocyanidin content. In this study, we investigated a dose effect (0, 0.01, 0.1, 1, 10, 50, and 100 μg/mL) of GSE and proanthocyanidin B2 (GSPB2) on the ROS content, cell proliferation, cell viability, and steroidogenesis in both primary luteinized granulosa cells (hGC) and the tumor granulosa cell line (KGN). The levels of ROS were measured using ROS-Glo assay. Cell proliferation and viability were evaluated by [3H]-thymidine incorporation and Cell Counting Kit-8 (CCK8) assay, respectively. Steroid secretion was evaluated by radioimmunoassay. We also analyzed the cell cycle component protein level and signaling pathways by immunoblot and the NOX4 mRNA expression by RTqPCR. From 0.1 to 1 μg/mL, GSE and GSBP2 reduced the ROS cell content and the NOX4 mRNA levels, whereas, GSE and GSBP2 increased the ROS cell content from 50 to 100 μM in both hGC and KGN. GSE and GSPB2 treatments at 50 and 100 μg/mL induced a delay in G1 to S phase cell cycle progression as determined by fluorescence-activated cell sorting. Consequently, they reduced cell growth, cyclin D2 amount, and Akt phosphorylation, and they increased protein levels of p21 and p27 cyclin-dependent kinase inhibitors. These data were also associated with an increase in cell death that could be due to a reduction in Bcl-2-associated death promoter (BAD) phosphorylation and an increase in the cleaved-caspase-3 level. All these negative effects were not observed at lower concentrations of GSE and GSPB2 (0.01 to 10 μg/mL). Interestingly, we found that GSE and GSPB2 treatments (0.1 to 100 μg/mL) improved progesterone and estradiol secretion and this was associated with a higher level of the cholesterol carriers, StAR (steroidogenic acute regulatory protein), CREB (Cyclic adenosine monophosphate Response Element-binding protein), and MAPK ERK1/2 (Mitogen-Activated Protein Kinases Extracellular signal-Regulated Kinases 1/2) phosphorylation in both hGC and KGN cells. Taken together, GSE and GSPB2 (0.1–10 μg/mL) in vitro treatments decrease oxidative stress and increase steroidogenesis without affecting cell proliferation and viability in human granulosa cells.