Inhibition Of Cyclin D1 Expression Research Articles

Crocin Potentiates Anti-tumor Properties of 5-FU by Regulating Cell Proliferation and Tumor Necrosis in Breast Cancer

Introduction:: Breast cancer is one of the most prevalent malignancies among women around the world. Crocus sativus, a natural food coloring and flavoring, has potent anti-tumor properties. The aim of the current study was to investigate the therapeutic potential of crocin, the main pharmacological active component of saffron, either alone or in combination with the standard chemotherapeutic treatment, 5-FU, in Breast cancer (BC) progression in both cellular and animal models. Material and Methods: MTT, Real-Time PCR, Western Blotting, Hematoxylin and eosin (H&E) tissue staining were applied to determine the anti-tumor properties of crocin in in vitro and in vivo samples. Results:: Our findings showed that crocin decreased breast cancer cell proliferation by suppressing cyclin D1 expression and Wnt/β-catenin signaling activation. Moreover, this molecule improved 5- FU anti-cancer activities by decreasing the tumor volume and weight, increasing tumor necrosis, and suppressing tumor inflammation in an animal model. Inflammation-associated anti-cancer activity of crocin is mediated by the down-regulation of pro-inflammatory genes, including IFN-γ and IL-1β, as well as inhibition of oxidative stress responses within the tumor environment. Conclusion:: This is the first study demonstrating the potent anti-tumor properties of crocin against BC progression. Our results suggest that this effective and low-toxic molecule could be a promising agent for reducing BC tumor progression when administered either alone or in combination with standard treatment in breast cancer patients.

Let-7c-5p associate with inhibition of phenobarbital-induced cell proliferation in human palate cells

Cleft palate (CP) is one of the most common congenital diseases, and is accompanied by a complicated etiology. Medical exposure in women is among one of the reasons leading to CP. Recently, it has been reported that microRNA (miRNA) plays a crucial role in palate formation and the disruption of miRNA that influence the development of CP. Although association with pharmaceuticals and miRNAs were suggested, it has remained largely unknow. The aim of the current investigation is to elucidate upon the miRNA associated with the inhibition of phenobarbital (PB)-induced cell proliferation in human embryonic palatal mesenchymal (HEPM) cells. We showed that PB inhibited HEPM cell viability in a dose-dependent manner. We demonstrated that PB treatment suppressed cyclin-D1 expression in HEPM cells. Furthermore, PB upregulated let-7c-5p expression and downregulated the expression of two downstream genes (BACH1 and PAX3). Finally, we demonstrated that the let-7c-5p inhibitor alleviated PB-induced inhibition of cell proliferation and altered BACH1 and PAX3 expression levels. These results suggest that PB suppresses cell viability by modulating let-7c-5p expression.

Curcumin Interferes with TGF- β 1-Induced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation.

To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis (RIF) progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18 (ADAMTS18) methylation. NRK-49F cells RIF model were induced with transforming growth factor β 1 (TGF- β 1). Effects of different concentrations of curcumin (0, 10, 20, and 30 μmol/L) on cell proliferation, cell cycle, cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8, flow cytometry and Western blot, respectively. ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction. ADAMTS18, fibronectin (FN), type I collagen (Col- I) and alpha-smooth muscle actin (α -SMA) mRNA and protein expressions were analyzed by real-time PCR (RT-PCR) and Western blot, respectively. Meanwhile, cells were treated with 50 mmol/L 5-aza-2'-deoxycytidine (5-aza-dC, demethylation agent) for 72 h. Effect of curcumin on extracellular matrix (ECM) deposition was evaluated by immunochemical staining and Western blot. NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control, ADAMTS18-knock-out (KO), and ADAMTS18-KO+ 30 μmol/L curcumin groups, and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated. Compared with the control group, TGF-β 1 significantly inhibited the proliferation of NRK-49F cells, blocked the G1/G0 phase, promoted cell apoptosis and inhibited cyclin D1 expression (P<0.01). Among the different concentrations of curcumin, 30 μmol/L curcumin significantly reversed these processes (P<0.01). Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN, Col- I and α-SMA (P<0.01). Curcumin and 5-zaz-dC had synergistic effects, promoting ADAMTS18 expression, removing ADAMTS18 methylation, and reducing ECM deposition. ADAMTS18 knockdown promoted ECM accumulation, and curcumin reversed this process (P<0.01). TGF-β 1-induced fibrosis in NRK-49F cells. Curcumin promoted ADAMTS18 expression, reduced ECM accumulation, and alleviated RIF progression by inhibiting ADAMTS18 methylation.

Cyclin D1 promotes radioresistance through regulation of RAD51 in melanoma.

Melanoma is a notoriously radioresistant type of skin cancer. Elucidation of the specific mechanisms underlying radioresistance is necessary to improve the clinical efficacy of radiation therapy. To identify the key factors contributing to radioresistance, five melanoma cell lines were selected for study and genes that were upregulated in relatively radioresistant melanomas compared with radiosensitive melanoma cells determined via RNA sequencing technology. In particular, we focused on cyclin D1 (CCND1), a well known cell cycle regulatory molecule. In radiosensitive melanoma, overexpression of cyclin D1 reduced apoptosis. In radioresistant melanoma cell lines, suppression of cyclin D1 with a specific inhibitor or siRNA increased apoptosis and decreased cell proliferation in 2D and 3D spheroid cultures. In addition, we observed increased expression of γ-H2AX, a molecular marker of DNA damage, even at a later time after γ-irradiation, under conditions of inhibition of cyclin D1, with a response pattern similar to that of radiosensitive SK-Mel5. In the same context, expression and nuclear foci formation of RAD51, a key enzyme for homologous recombination (HR), were reduced upon inhibition of cyclin D1. Downregulation of RAD51 also reduced cell survival to irradiation. Overall, suppression of cyclin D1 expression or function led to reduced radiation-induced DNA damage response (DDR) and triggered cell death. Our collective findings indicate that the presence of increased cyclin D1 potentially contributes to the development of radioresistance through effects on RAD51 in melanoma and could therefore serve as a therapeutic target for improving the efficacy of radiation therapy.

Glucosamine-Modified Reduction-Responsive Polymeric Micelles for Liver Cancer Therapy.

In this work, glucose transporter-1 (GLUT-1) and glutathione (GSH) over-expression in liver cancer was utilized to design a reduction-responsive and active targeting drug delivery system AG-PEG-SS-PCL (APSP) for the delivery of sorafenib (SF). The SF-APSP micelles were prepared using the thin film hydration method and characterized by various techniques. In vitro release experiments showed that the cumulative release of SF-APSP micelles in the simulated tumor microenvironment (pH 7.4 with GSH) reached 94.76 ± 1.78% at 48 h, while it was only 20.32 ± 1.67% in the normal physiological environment (pH 7.4 without GSH). The in vitro study revealed that glucosamine (AG) enhanced the antitumor effects of SF, and SF-APSP micelles inhibited proliferation by targeting HepG2 cells and suppressing cyclin D1 expression. The in vivo antitumor efficacy study further confirmed that the SF-APSP micelles had excellent antitumor effects and better tolerance against nude mouse with HepG2 cells than other treatment groups. All in all, these results indicated that SF-APSP micelles could be a promising drug delivery system for anti-hepatoma treatment.

Designing Tailored Thiosemicarbazones with Bespoke Properties: The Styrene Moiety Imparts Potent Activity, Inhibits Heme Center Oxidation, and Results in a Novel "Stealth Zinc(II) Complex".

A novel, potent, and selective antitumor agent, namely (E)-3-phenyl-1-(2-pyridinyl)-2-propen-1-one 4,4-dimethyl-3-thiosemicarbazone (PPP44mT), and its analogues were synthesized and characterized and displayed strikingly distinctive properties. This activity was mediated by the inclusion of a styrene moiety, which through steric and electrochemical mechanisms prevented deleterious oxy-myoglobin or oxy-hemoglobin oxidation relative to other potent thiosemicarbazones, i.e., di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Structure-activity relationship analysis demonstrated specific tuning of PPP44mT electrochemistry further inhibited oxy-myoglobin or oxy-hemoglobin oxidation. Both PPP44mT and its Cu(II) complexes showed conspicuous almost immediate cytotoxicity against SK-N-MC tumor cells (within 3 h). In contrast, [Zn(PPP44mT)2] demonstrated a pronounced delay in activity, taking 48 h before marked antiproliferative efficacy was apparent. As such, [Zn(PPP44mT)2] was designated as a "stealth Zn(II) complex" that overcomes the near immediate cytotoxicity of PPP44mT or its copper complexes. Upon examination of the suppression of oncogenic signaling, [Zn(PPP44mT)2] was superior at inhibiting cyclin D1 expression compared to DpC or Dp44mT.

CHREBP suppresses gastric cancer progression via the cyclin D1-Rb-E2F1 pathway

Accumulating evidence has demonstrated that carbohydrate response element binding protein (CHREBP) has a crucial function in tumor pathology. In this study, we found CHREBP downregulation in gastric cancer (GC) tissues, and CHREBP was determined to be an independent diagnostic marker of GC. The downregulation of CHREBP promoted cell proliferation and inhibited apoptosis. Moreover, the level of cyclin D1 was significantly correlated with CHREBP expression in GC and paracancerous normal samples. In addition, CHREBP transcriptionally inhibited cyclin D1 expression in GC cells. Tumor suppressor activity of CHREBP could be affected by the upregulation of cyclin D1. In summary, CHREBP was found to be an independent diagnostic marker of GC and to influence GC growth and apoptosis via targeting the cyclin D1-Rb-E2F1 pathway.

The Effect of Avidin on Viability and Proliferation of Colorectal Cancer Cells HT-29.

Objective:The aim of this study was to analyze the effect of avidin treatment on cell viability, proliferation and cyclin D1 expression in colorectal cancer cells HT-29. Methods:Colorectal cancer cell line HT-29 incubated with 50, 100, 150, and 200 μg/mL of avidin concentration during 24, 48, and 72 hours, then the cell viability and proliferation were analyzed. Each avidin concentration was conducted together with HT-29 cell line without avidin treatment as a control group. The cell viability was measured by MTS assay and the proliferation was measured by BrdU (5-bromo-2′-deoxyuridine) cell proliferation assay. According to cell viability and proliferation result, we determined the 100 μg/mL avidin concentration for analyzing mRNA and protein of cyclin D1. Results:We demonstrated that the viability and proliferation of HT-29 cells were significantly decreased in all concentration of avidin treatment compared to control. The cell proliferation showed larger reduction in avidin treatment rather than cell viability. This proves avidin could inhibit proliferation of colorectal cancer cell HT-29 quite well. The expression of cyclin D1, both mRNA and protein, was also significantly decreased after the avidin treatment group compared to control group, it supports the suppression of proliferation result. Conclusion:We concluded that avidin treatment could decrease cell viability and proliferation, accompanied by suppression of cyclin D1 expression in colorectal cells HT-29.

CAMSAP3 depletion induces lung cancer cell senescence-associated phenotypes through extracellular signal-regulated kinase inactivation.

BackgroundCellular senescence is an aging‐related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin‐regulated spectrin‐associated protein 3 (CAMSAP3), a minus‐end microtubule‐stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood.MethodsThe function of CAMSAP3 on the regulation of cellular senescence‐associated phenotypes in human non‐small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence‐associated phenotypes were observed by SA‐β‐Gal staining. Quantitative RT‐PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3‐ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed.Results CAMSAP3 knockout promotes lung cancer cell senescence‐associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p‐ERK) was markedly downregulated in CAMSAP3‐deleted cells, suppressing cyclin D1 expression levels, and full‐length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3‐modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p‐ERK and cyclin D1, resulting in a senescence‐like phenotype.ConclusionThis study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence‐associated phenotypes via the modulation of p‐ERK/cyclin D1 signaling.

The Role of Egln2/Proline Hydroxylase 1 in Regulation of Cyclin D1 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the CCND1 gene (which encodes for cyclin D1) on chromosome 11q13 and an immunoglobulin heavy chain gene promoter on chromosome 14q32. Despite a high rate of complete remission (response 20-80%) by many chemotherapy regimens, MCL typically relapses after treatment, with a median survival time of approximately 3-4 years. Novel therapeutic approaches target several cellular pathways of MCL (CDK, proteasome, mTOR, or NFκB inhibitors and others) but so far have shown only modest effects on overall survival.Iron is essential for cell proliferation. Although iron chelators were developed for treatment of iron overload diseases, depletion by chelators leads to cancer cell cycle arrest and apoptosis. Iron chelation directly or indirectly affects multiple proteins controlling DNA replication, DNA repair, cell cycle progression and multiple metabolic processes; one known target of iron chelation is cyclin D1. Besides iron chelation-induced suppression of CCND1 gene transcription, Tjendraputra, et al (Blood, 2007), postulated that iron chelation causes posttranslational degradation of cyclin D1 via von Hippel-Lindau protein-independent ubiquitination and subsequent proteasomal degradation.We hypothesized that iron chelation would be particularly effective in MCL with upregulated cyclin D1. We treated human MCL cell lines (Jeko-1, Mino and HBL-2) with 250 µM deferoxamine mesylate (DFO), which caused decreased cell growth, increased apoptosis, and decreased cyclin D1 mRNA and protein levels. These cell lines were more susceptible to treatment with DFO than B-cell lymphoma derived cell lines without constitutively active cyclin D1 (SUDHL-6, DG-75). A possible cytotoxic effect due to a high concentration of DFO was ruled out by abrogating the DFO effect by concomitant administration of ferric ammonium citrate.We further investigated the molecular mechanism underlying decreased cyclin D1 mRNA and protein levels in MCL cell lines after DFO treatment. We tested whether the DFO effect may be due to inhibition of one of the iron-dependent hypoxia-inducible factor (HIF) hydroxylases. Expression analysis of several hypoxia related genes in MCL cell lines treated with DFO revealed down-regulation of PHD1 encoded by the EglN2 gene, a member of the PHD family. All members of the PHD protein family contribute to regulation of HIF, but only EglN1 gene encoding PHD2 has a significant role in oxygen sensing under physiological conditions. EglN2 /PHD1 and EglN3 /PHD3 also have HIF-independent functions in control of cell proliferation and apoptosis. In breast cancer, a loss of EglN2 /PHD1 leads to down-regulation of cyclin D1 and decreases cell proliferation in a HIF-independent manner (Zhang et al, Cell, 2009). To further confirm that EglN2 /PHD1 inhibition is linked to suppression of cyclin D1, we treated MCL cell lines with known PHD inhibitors, 1 mM DMOG and 50 µM FG-4497. Proliferation was markedly reduced and down-regulation of cyclin D1 was confirmed in mRNA and protein levels. It has been proposed that the EglN 2/PHD1 substrate linked to repression of cyclin D1 is the transcription factor FOXO3a. An inability of EglN2 /PHD1 to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses cyclin D1 expression by a yet unknown mechanism (Zheng X et al, Genes & Development, 2014). We measured FOXO3a expression levels in MCL cell lines after treatment with DFO and DMOG and found it significantly upregulated. However, knock-down of FOXO3a by CRISPR/Cas9 technology in MCL line Mino did not affect cyclin D1 levels after DFO treatment. These data suggest that in MCL cell lines treated with DFO, accumulation of FOXO3a is present, but not required for cyclin D1 repression and that cyclin D1 down-regulation mediated by inhibition of PHD1 is a result of another, as yet unknown mechanism. Thus, our preliminary data unveiled new insights into regulation of cyclin D1 in MCL and opens new possibilities for MCL targeted therapy with chelation therapy and/or PHD inhibitors.This study was supported by research funding from Czech Science Foundation, project GACR 15-18046Y (OB, LL), by Ministry of Health Czech Republic, grant AZV 16-31689A (VD) and from the Ministry of Education, Youth and Sports, Czech Republic, Program KONTAKT II (LH15223) (LL, KK, VD). DisclosuresNo relevant conflicts of interest to declare.

Effects of Xinwei granule on expression levels of cyclin D1 and its upstream genes in gastric intraepithelial neoplasia tissues

Purpose: To explore the effects of Xinwei granule (XWG) on low-grade gastric intraepithelial neoplasia (LGIN) and the underlying mechanisms.&#x0D; Methods: To establish LGIN model, Wistar rats were treated with N-methyl-N'-nitrosoguanidine for 3 months. LGIN model rats were randomly grouped into five groups (n = 15), viz, negative control (NC), normal saline (NS) group, Xinwei granule (XWG) group, Weifuchun tablet (WFCT) group, and vatacoenayme tablet (VT) group. Normal rats (n = 17) served as negative control. Histological evaluation of gastric mucosa was undertaken using hematoxylin and eosin staining. Quantitative realtime polymerase chain reaction (qRT-PCR), western blot, and immunohistochemical assays were performed to determine mRNA expressions, protein expression, and the distribution of cyclin D1, kruppel-like factor 4 (KLF4), and p21-WAF1-CIP1, respectively.&#x0D; Results: Compared with LGIN group, the body weight of the rats increased in XWG, WFCT, and VT groups. The pathological characteristics of LGIN group were alleviated by XWG, WFCT and VT treatments. The positive expression of cyclin D1 was enhanced in LGIN group, but reduced in XWG, WFCT and VT groups. The expression levels of KLF4 and p21-WAF1-CIP1, upstream regulators of cyclin D1 reduced in LGIN groups. However, administration of XWG, WFCT and VT strengthened the expressions of KLF4 and p21-WAF1-CIP1. More importantly, the protective effects of XWG against LGIN were superior to those of WFCT and VT.&#x0D; Conclusion: Xinwei granules alleviate LGIN in vivo by inhibiting cyclin D1 expression and enhancing KLF4 and p21-WAF1-CIP1 expression.

Identification of a druggable protein–protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1

The TP53 gene is the most frequently mutated gene in human cancers, and the majority of TP53 mutations are missense mutations. As a result, these mutant p53 (mutp53) either directly lose wildtype p53 (wtp53) tumor suppressor function or exhibit a dominant negative effect over wtp53. In addition, some mutp53 have acquired new oncogenic function (gain of function). Therefore, targeting mutp53 for its degradation may serve as a promising strategy for cancer prevention and therapy. Based on our previous finding that farnesylated DNAJA1 is a crucial chaperone in maintaining mutp53 stabilization, and by using an in silico approach, we built 3D homology models of human DNAJA1 and mutp53R175H proteins, identified the interacting pocket in the DNAJA1–mutp53R175H complex, and found one critical druggable small molecule binding site in the DNAJA1 glycine/phenylalanine-rich region. We confirmed that the interacting pocket in the DNAJA1–mutp53R175H complex was crucial for stabilizing mutp53R175H using a site-directed mutagenesis approach. We further screened a drug-like library to identify a promising small molecule hit (GY1-22) against the interacting pocket in the DNAJA1–mutp53R175H complex. The GY1-22 compound displayed an effective activity against the DNAJA1–mutp53R175H complex. Treatment with GY1-22 significantly reduced mutp53 protein levels, enhanced Waf1p21 expression, suppressed cyclin D1 expression, and inhibited mutp53-driven pancreatic cancer growth both in vitro and in vivo. Together, our results indicate that the interacting pocket in the DNAJA1–mutp53R175H complex is critical for mutp53’s stability and oncogenic function, and DNAJA1 is a robust therapeutic target for developing the efficient small molecule inhibitors against oncogenic mutp53.

Sasa veitchii extract induces anticancer effects via inhibition of cyclin D1 expression in MCF-7 cells.

ABSTRACT Sasa veitchii and other Sasa species are traditional medicinal herbs belonging to a group of Japanese bamboos collectively called Kumazasa, and these species possess the potential for a wide variety of uses. The present study aimed to elucidate the anticancer mechanisms exerted by S. veitchii extract (SE) against a human breast cancer cell line, MCF-7 cells. Freeze-dried Sunchlon® was used as the SE, and cell proliferation activity was measured using the [3H]-thymidine incorporation assay. Induction of apoptosis was assessed via Annexin V and caspase-3 fluorescent staining, the induction of necrosis was measured via propidium iodide staining, and cell cycle-related protein expression was determined using western blotting. The IC50 value of the SE was 7.7 μg/mL in MCF-7 cells. Although the primary active ingredient in Sunchlon® is sodium copper chlorophyllin (0.25%), the present results indicated that ingredients other than SCC exert anti-cancer activities (the IC50 value of SCC was 715 μg/mL), and late apoptosis or necrosis was induced in an SE dose-dependent manner. The expression levels of cyclin D1 and Cdk6 were decreased after SE treatment, and there was no change in the Cdk1/2 expression levels. Additionally, the expression of the necrosis-related cell death indicators RIP1 and RIP3 was increased in response to high-dose SE treatments, and this was indicative of cells preparing for programmed cell death. SE induces cell death in MCF-7 cells via the inhibition of cyclin D1 expression at low concentrations, and this extract induces programmed necrosis (necroptosis) by potentiating RIP1/RIP3 expression.

NLRP6 suppresses gastric cancer growth via GRP78 ubiquitination

Nod-like receptor pyrin domain-containing protein 6 (NLRP6) plays a key role in innate immunity, host defense and tumorigenesis. Our previous study has demonstrated the tumor suppressor role of NLRP6 in gastric cancer. In the present study, we explored the interaction protein of NLRP6 by Flag-tagged immunoprecipitation assay and liquid chromatography/mass spectrometry-based proteomics analysis. The 78 kDa glucose-regulated protein (GRP78), a heat shock protein, was identified as an interaction protein of NLRP6. The binding of NLRP6 to GRP78 was through the Pyrin domain, and the substrate binding domain (SBD) domain of GRP78 was responsible for the interaction with NLRP6. NLRP6 overexpression enhanced the polyubiquitination of GRP78 in gastric cancer cells. Overexpression of GRP78 abolished the effects of NLRP6 overexpression in gastric cancer cell proliferation, cell cycle progression, cell apoptosis, migration and Cyclin D1 expression. GRP78 knockdown reversed the effects of NLRP6 knockdown on cell proliferation and cell cycle progression. NLRP6 expression was negatively correlated with GRP78 expression in human gastric tissues. Tumorigenicity assay indicated that GRP78 mediated the functions of NLRP6 on gastric cancer cell growth in vivo. ON-013100, which could inhibit Cyclin D1 expression, was less effective in treating xenografts of gastric cancer cells with higher level of NLRP6 than in those with lower level of NLRP6. In conclusion, our study suggested that NLRP6 exerted inhibitory effects on gastric cancer cell growth by promoting the ubiquitination of GRP78.

Methylation-associated silencing of miR-193b improves the radiotherapy sensitivity of esophageal cancer cells by targeting cyclin D1 in areas with zinc deficiency

BackgroundAlthough radiotherapy is an important treatment mode for esophageal cancer (EC), the outcome remains unsatisfactory due to radioresistance, and the key cause of radiotherapy resistance is a change in the cell cycle. Zinc deficiency (ZD) has a significant influence on the cell cycle, and this effect is a common phenomenon in areas with a high incidence of esophageal cancer. MethodsRadioresistant sub-cell lines were established by exposing esophageal cancer cells to nine rounds of X-ray irradiation at a dose of 2 Gy. The cells were treated with a range of different concentrations of zinc and overexpression of miR-193b. And proliferation, colony formation, cell cycle and apoptosis assays were then conducted. Luciferase reporter assays were performed to confirm direct interactions between miR-193b and ZRT/IRT-like protein 5 (ZIP5) and between miR-193b and Cyclin D1. Analysis of clinical and follow-up data was performed using data obtained from 75 patients from Cixian, a well-known high incidence area of esophageal cancer. All these patients are unable to tolerate surgery due to their advanced age or advanced stage, and serum specimens were obtained before the patients received therapy. ResultsThe cell cycle of radioresistant cells is blocked in G0/G1 phase (from 50% to 68%). The expression level of Cyclin D1 was decreased and miR-193b was increased in radioresistant cells (P < 0.001). ZD decreased the proportion of cells in G0/G1 phase both in EC (from 50% to 32%) and radioresistant (from 68% to 47%) cells. And the radioresistance of these two cells were decreased. ZD increased the expression of Cyclin D1 (P < 0.001) and inhibited the level of miR-193b (P < 0.001). Up-regulation of miR-193b recovered the proportion of cells in G0/G1 phase and the radioresistance, meanwhile, recovered the altered expression levels of Cyclin D1 and miR-193b of these two cells by ZD. ZD enhanced DNMT activity both in EC (32%) and radioresistant (26%). And miR-193b was hypermethylated both in EC and radioresistant cells. MiR-193b suppressed Cyclin D1 expression by targeting the 3′UTR of Cyclin D1 mRNA. The expression level of miR-193b in the serum of patients was correlated with the disease control rate (DCR) and had a good diagnostic value for distinguishing DCR of EC patients (AUC = 0.710, 95%CI: 0.580–0.839, P = 0.003). And the level of miR-193b was correlated with overall survival (OS) (HR = 0.208, 95%CI: 0.094–0.464). ConclusionsThe methylation-mediated silencing of miR-193b in EC cells due to ZD increased the expression of ZIP5, and the overexpression of ZIP5 increased the intracellular zinc levels to maintain zinc homeostasis. Meanwhile, the silencing of miR-193b increased the sensitivity of radiotherapy by promoting the expression of Cyclin D1.

Activation of metabotropic glutamate receptor 4 regulates proliferation and neural differentiation in neural stem/progenitor cells of the rat subventricular zone and increases phosphatase and tensin homolog protein expression.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III β-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.

Pregnancy facilitates maternal liver regeneration after partial hepatectomy.

Liver resection induces robust liver regrowth or regeneration to compensate for the lost tissue mass. In a clinical setting, pregnant women may need liver resection without terminating pregnancy in some cases. However, how pregnancy affects maternal liver regeneration remains elusive. We performed 70% partial hepatectomy (PH) in nonpregnant mice and gestation day 14 mice, and histologically and molecularly compared their liver regrowth during the next 4 days. We found that compared with the nonpregnant state, pregnancy altered the molecular programs driving hepatocyte replication, indicated by enhanced activities of epidermal growth factor receptor and STAT5A, reduced activities of cMet and p70S6K, decreased production of IL-6, TNFα, and hepatocyte growth factor, suppressed cyclin D1 expression, increased cyclin A1 expression, and early activated cyclin A2 expression. As a result, pregnancy allowed the remnant hepatocytes to enter the cell cycle at least 12 h earlier, increased hepatic fat accumulation, and enhanced hepatocyte mitosis. Consequently, pregnancy ameliorated maternal liver regeneration following PH. In addition, a report showed that maternal liver regrowth after PH is driven mainly by hepatocyte hypertrophy rather than hyperplasia during the second half of gestation in young adult mice. In contrast, we demonstrate that maternal liver relies mainly on hepatocyte hyperplasia instead of hypertrophy to restore the lost mass after PH. Overall, we demonstrate that pregnancy facilitates maternal liver regeneration likely via triggering an early onset of hepatocyte replication, accumulating excessive liver fat, and promoting hepatocyte mitosis. The results from our current studies enable us to gain more insights into how maternal liver regeneration progresses during gestation.NEW & NOTEWORTHY We demonstrate that pregnancy may generate positive effects on maternal liver regeneration following partial hepatectomy, which are manifested by early entry of the cell cycle of remnant hepatocytes, increased hepatic fat accumulation, enhanced hepatocyte mitosis, and overall accelerated liver regrowth.

Wogonin inhibits cell cycle progression by activating the glycogen synthase kinase-3 beta in hepatocellular carcinoma

BackgroundWogonin has been reported to exhibit various biological activities such as anti-inflammation, anti-microbial, and anti-tumor. Previous studies have demonstrated that wogonin could down-regulate Cyclin D1 activity on multiple cancers. However, the related mechanisms have not been fully elucidated so far. PurposeThe aim of the current study was to explore whether wogonin can suppress hepatocellular carcinoma (HCC) progression and the mechanism of wogonin in inhibiting Cyclin D1 expression. MethodsHerein, we assessed the anti-tumor activity of wogonin against hepatocellular carcinoma (HCC) by MTT assay, clonogenic assay, cell cycle analysis and orthotopic xenograft mouse models. Western blot, immunofluoscence assay, co-immunoprecipitation assay, docking program, surface plasmon resonance, site-directed mutagenesis assay and immunohistochemical assay were performed for exploring the underlying mechanisms of wogonin-induced growth inhibition in HCC. ResultsOur results showed that non-toxic dosage of wogonin (10, 20 µM) could inhibit cells proliferation and suppress cells cycle progression in MHCC97L and HepG2 cell. Moreover, the findings from the western blot and immunofluoscence assay confirmed the inhibition action of wogonin (10, 20 µM) on Cyclin D1 expression in MHCC97L cells, and wogonin (10, 20 µM) pre-treatment was capable of promoting Cyclin D1 ubiquitination and degradation in MHCC97L cell. In addition, wogonin promoted phosphorylation of Cyclin D1 on threonine-286 site, the mutation of threonine-286 to alanine-286A blocked Cyclin D1 proteolysis induced by wogonin. Wogonin-promoted Cyclin D1 phosphorylation and subsequent proteolysis may associate with the activation of GSK3beta in cancer cells. The phosphorylated form of GSK3beta (active form) expression was significantly increased after wogonin (20 µM) exposure. Molecular docking study and Biacore SPR analysis of GSK3beta mutant further validated the high-affinity wogonin binding site on GSK3beta. Moreover, in vivo studies further confirmed that phospho-GSK3beta Tyr216 was over-expressed in HCC specimens after wogonin treatment while the amount of Cyclin D1 was significantly decreased. ConclusionIn summary, our data reveal a novel molecular mechanism by which wogonin induces HCC cells cycle arrest and suppresses tumor proliferation.

JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle

JARID2 is a noncatalytic member of the polycomb repressive complex 2 (PRC2) which methylates of histone 3 lysine 27 (H3K27). In this work, we show that JARID2 and the PRC2 complex regulate the cell cycle in skeletal muscle cells to control proliferation and mitotic exit. We found that the stable depletion of JARID2 leads to increased proliferation and cell accumulation in S phase. The regulation of the cell cycle by JARID2 is mediated by direct repression of both cyclin D1 and cyclin E1, both of which are targets of PRC2-mediated H3K27 methylation. Intriguingly, we also find that the retinoblastoma protein (RB1) is a direct target of JARID2 and the PRC2 complex. The depletion of JARID2 is not sufficient to activate RB1. However, the ectopic expression of RB1 can suppress cyclin D1 expression in JARID2-depleted cells. Transient depletion of JARID2 in skeletal muscle cells leads to a transient up-regulation of cyclin D1 that is quickly suppressed with no resulting effect on proliferation, Taken together, we show that JARID2 and the PRC2 complex regulate skeletal muscle proliferation in a precise manner that involves the repression of cyclin D1, thus restraining proliferation and repressing RB1, which is required for mitotic exit and terminal differentiation.

DNA Methyltransferase 1 Affects Proliferation and Migration of Colorectal Cancer Cells by Regulating Ras Association Domain Family 1 Methylation

The effects of DNMT1 on human colorectal cancer cell proliferation and apoptosis as well as the possible underlying mechanisms were investigated. SW480 cells were treated with the DNA methyltransferase inhibitor 5-AZA-CdR. qRT-PCR was used to analyze mRNA expression, whereas protein expression was evaluated by western blotting. Cell growth, migration, and invasion were measured by MTT, transwell migration, and wound healing assays, respectively. Cell morphology was observed using hematoxylin and eosin (H&amp;E) staining. 5-AZA-CdR treatment inhibited DNMT1 expression in colorectal cancer cells. Further, low expression of DNMT1 regulated RASSF1A-mediated inhibition of CyclinD1 expression, as well as colon cancer cell morphology, proliferation, and migration via RASSF1A methylation. Our results provide a basis for therapeutic strategies based on DNMT1 and RASSF1A for colorectal cancer.