Cyclin CDK4 Research Articles

Oxymatrine induces cell cycle arrest and apoptosis and suppresses the invasion of human glioblastoma cells through the EGFR/PI3K/Akt/mTOR signaling pathway and STAT3.

Oxymatrine (OM), a natural quinolizidine alkaloid extracted from the traditional Chinese herb Sophora flavescens, has been revealed to produce antitumor activities in various cancer cell lines, including glioblastoma lines, invitro. However, the mechanisms by which OM exerts its antitumor effect against glioma are poorly understood. The aim of this study was to investigate the role of OM in the proliferation, apoptosis and invasion of glioma cells and to reveal the underlying mechanisms. The effects of OM on U251MG cells invitro were determined using a Cell Counting Kit‑8 (CCK‑8) assay, flow cytometric analysis, AnnexinV‑FITC/PI staining, DAPI staining, a terminal deoxynucleotidyl transferase‑mediated dUTP nick end‑labeling (TUNEL) assay, a Transwell assay and western blotting. Our data indicated that OM inhibited proliferation, arrested the cell cycle at the G0/G1phase, decreased the expression levels of G1 cell cycle regulatory proteins (cyclinD1, CDK4 and CDK6), inhibited invasion and induced apoptosis in glioma cells. Additional investigations revealed that the expression levels of p‑STAT3 and key proteins in the EGFR/PI3K/Akt/mTOR signaling pathway, such as p‑EGFR, p‑Akt and p‑mTOR, were markedly decreased after OM treatment, while the total STAT3, EGFR, Akt and mTOR levels were not affected. These findings indicated that the EGFR/PI3K/Akt/mTOR signaling pathway and STAT3 suppression may be a potential mechanism of the OM‑mediated antitumor effect in glioblastoma cells and that EGFR may be a target of OM. Hence, OM may be a promising drug and may offer a novel therapeutic strategy for malignant gliomas in the future.

The poly (ADP-ribose) polymerase inhibitor rucaparib suppresses proliferation and serves as an effective radiosensitizer in cervical cancer.

Objectives Our goal was to investigate the effects of rucaparib on the proliferation of cervical cancer cells and sensitivity to radiotherapy. Methods We used the human cervical cancer cell lines Hela and Siha and evaluated their viability and activity using various methods. Cellular proliferation was assessed by CCK-8 and clonogenic assays after treatment with rucaparib. Cell cycle analysis was performed using propidium iodide staining. Western immunoblotting analysis was used to detect the expression of cyclin D1 and CDK4. Immunofluorescence staining assay was performed to detect the expression of the DNA injury marker ץ-H2AX after treatment with rucaparib and radiotherapy. Animal experiments were also performed to evaluate tumor size after treatment with rucaparib. Immunohistochemistry was performed to analyze the expression of Ki-67. Results Rucaparib suppressed proliferation, induced G2/M phase arrest, and reduced the expression of cyclin D1 and CDK4 in cervical cancer cells. When rucaparib was combined with radiotherapy in cervical cancer cells, clone formation decreased significantly and G2/M phase arrest was accentuated. The expression of the DNA-damage marker ץ-H2AX was increased significantly, and rucaparib suppressed tumor growth in vivo. Conclusions Rucaparib exerts significant anti-proliferative effects and can serve as an effective radiosensitizer in cervical cancer, suggesting its candidacy in cervical cancer treatment and worthiness for further investigation.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle–Related Gene Transcription

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

Cyclin A Turns on Bora to Light the Path to Mitosis

Mitotic entry is an irreversible cell-cycle transition that requires the robust, rapid activation of cyclin-dependent kinase 1 (Cdk1). In this issue of Developmental Cell, Vigneron etal. (2018) identify cyclin A-Cdk1 as the factor triggering mitotic entry through the phosphorylation of Bora, an activator of the kinase Aurora A.

Schisantherin A Improves Learning and Memory of Mice with D-Galactose-Induced Learning and Memory Impairment Through Its Antioxidation and Regulation ofp19/p53/p21/Cyclin D1/CDK4/RBGene Expressions

Schisantherin A (SCA) was evaluated for possible function in restoring the learning and memory impairment induced by D-galactose in mice. ICR mice were treated with D-galactose subcutaneously (220 mg·kg-1), and followed by SCA in different doses (1.25, 2.50 and 5.00 mg·kg-1, administered orally) for 42 days. Effects of SCA on learning and memory were examined by step-through tests and Morris water maze tests. The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) in the peripheral blood and hippocampus of mice were assayed by water-soluble tetrazolium-1 (WST-1) and thiobarbituric acid (TBA) methods. The contents of 8 hydroxy deoxy guanosine (8-OHdG) in the hippocampus of mice were detected by immunosorbent assay methods, respectively. Quantitative real-time PCR and Western Blot were respectively used to detect the expression of p19, p53, p21, cyclin D1, CDK4 and RB genes, and the phosphorylation of RB in the hippocampus of mice. We found that SCA significantly improved the learning and memory impairment induced by D-galactose in mice. After SCA treatment, SOD activity was increased and the content of MDA was decreased in both peripheral blood and hippocampus of mice. 8-OHDG content was also decreased in the hippocampus of mice. Furthermore, the expression of p19, p53 and p21 genes was reduced and the expression of cyclin D1 and CDK4 and the phosphorylation of RB protein were elevated in the hippocampus. SCA may improve the learning and memory impairment induced by D-galactose by enhancing the antioxidant capacity, and regulating the expression of p19/p53/p21/cyclinD1/CDK4 genes, and the phosphorylation of RB protein in the hippocampus of mice.

Oleanolic acid induces p53-dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in prostatic cancer xenografts in mice.

We evaluated oleanolic acid (OA)-induced anti-cancer activity, apoptotic mechanism, cell cycle status, and MAPK kinase signaling in DU145 (prostate cancer), MCF-7 (breast cancer), U87 (human glioblastoma), normal murine liver cell (BNL CL.2) and human foreskin fibroblast cell lines (Hs 68). The IC50 values for OA-induced cytotoxicity were 112.57 in DU145, 132.29 in MCF-7, and 163.60 in U87 cells, respectively. OA did not exhibit toxicity in BNL CL. 2 and Hs 68 cell lines in our experiments. OA, at 100 µg/mL, increased the number of apoptotic cells to 27.0% in DU145, 27.0% in MCF-7, and 15.7% in U87, when compared to control cells. This enhanced apoptosis was due to increases in p53, cytochrome c, Bax, PARP-1 and caspase-3 expression in DU145, MCF-7 and U87 cell lines. OA-treated DU145 cells were arrested in G2 because of the activation of p-AKT, p-JNK, p21 and p27, and the decrease in p-ERK, cyclin B1 and CDK2 expression; OA-treated MCF-7 cells were arrested in G1 owing to the activation of p-JNK, p-ERK, p21, and p27, and the decrease in p-AKT, cyclin D1, CDK4, cyclin E, and CDK2; and OA-treated U87 cells also exhibited G1 phase arrest caused by the increase in p-ERK, p-JNK, p-AKT, p21, and p27, and the decrease in cyclin D1, CDK4, cyclin E and CDK2. Thus, OA arrested the cell cycle at different phases and induced apoptosis in cancer cells. These results suggested that OA possibly altered the expression of the cell cycle regulatory proteins differently in varying types of cancer.

Hydrogen gas inhibits lung cancer progression through targeting SMC3

Lung cancer is one of the most common lethal malignancies in the globe. The patients’ prognoses are dim due to its high metastatic potential and drug resistance. Therefore, in the present study, we aim to find a more potent therapeutic approach for lung cancer. We mainly explored the function of hydrogen gas (H2) on cell viability, apoptosis, migration and invasion in lung cancer cell lines A549 and H1975 by CCK-8, flow cytometry, wound healing and transwell assays, respectively. We used RNA-seq, qPCR and western blotting to detect the different expression genes (DEGs) between H2 group and control group to find the gene related to chromosome condensation. Besides, we confirmed the structural maintenance of chromosomes 3 (SMC3) and H2 on the progression of lung cancer in vitro and vivo. Results showed that H2 inhibited cell viability, migration and invasion, and catalyzed cell apoptosis and H2 induced A549 and H1975 cells G2/M arrest. Besides, H2 down-regulated the expression of NIBPL, SMC3, SMC5 and SMC6, and also reduced the expression of Cyclin D1, CDK4 and CDK6. H2 translocated the subcellular location of SMC3 during cell division and decreased its stability and increased its ubiquitination in both A549 and H1975 cells. In addition, inhibition of the proliferation, migration and invasion and promotion of the apoptosis of A549 and H1975 cells induced by H2 were all abolished when overexpressed SMC3 in the presence of H2. Animal experimental assay demonstrated that the tumor weight in H2 group was significantly smaller than that in control group, but was bigger than cis-platinum group. The expression of Ki-67, VEGF and SMC3 were decreased when mice were treated with H2 or cis-platinum, especially for cis-platinum. All data suggested that H2 inhibited lung cancer progression through down-regulating SMC3, a regulator for chromosome condensation, which provided a new method for the treatment of lung cancer.

LncRNA ABHD11-AS1 promotes the development of endometrial carcinoma by targeting cyclin D1.

To investigate the expression, role and mechanism of action of long non‐coding RNA (lncRNA) ABHD11‐AS1 in endometrial carcinoma. The expression of lncRNA ABHD11‐AS1 was quantified by qRT‐PCR in human endometrial carcinoma (n = 89) and normal endometrial tissues (n = 27). LncRNA ABHD11‐AS1 was stably overexpressed or knocked‐down in endometrial carcinoma cell lines to examine the cellular phenotype and expression of related molecules. Compared to normal endometrial tissue, lncRNA ABHD11‐AS1 was significantly overexpressed in endometrial carcinoma. Overexpression of lncRNA ABHD11‐AS1 promoted the proliferation, G1‐S progression, invasion and migration of endometrial cancer cells; inhibited apoptosis; up‐regulated cyclin D1, CDK1, CDK2, CDK4, Bcl‐xl and VEGFA; and down‐regulated p16, while ABHD11‐AS1 down‐regulation has the opposite effect. RNA pull down demonstrated that lncRNA ABHD11‐AS1 binds directly to cyclin D1. Knockdown of cyclin D1 can reverse the effect of ABHD11‐AS1. Overexpression of lncRNA ABHD11‐AS1 increased the tumorigenicity and up‐regulated cyclin D1 in an in vivo model of endometrial cancer in nude mice. LncRNA ABHD11‐AS1 functions as an oncogene to promote cell proliferation and invasion in endometrial carcinoma by positively targeting cyclin D1.

Synchronous and Time-Dependent Expression of Cyclins, Cyclin-Dependant Kinases, and Apoptotic Genes in the Rumen Epithelia of Butyrate-Infused Goats.

In our previous study, we demonstrated that butyrate induced ruminal epithelial growth through cyclin D1 upregulation. Here, we investigated the influence of butyrate on the expression of genes associated with cell cycle and apoptosis in rumen epithelium. Goats (n = 24) were given an intra ruminal infusion of sodium butyrate at 0.3 (group B, n = 12) or 0 (group A, n = 12) g/kg of body weight (BW) per day before morning feeding for 28 days and were slaughtered (4 goat/group) at 5,7 and 9 h after butyrate infusion. Rumen fluid was analyzed for short chain fatty acids (SCFAs) concentration. Ruminal tissues were analyzed for morpho-histrometry and the expressions of genes associated with cell cycle and apoptosis. The results revealed that the ruminal butyrate concentration increased (P < 0.05) in B compared to group A. Morphometric analysis showed increased (P < 0.05) papillae size associated with higher number of cell layers in epithelial strata in B compared to A. Butyrate-induced papillae enlargement was coupled with enhanced mRNA expression levels (P < 0.05) of cyclin D1, CDK2, CDK4, and CDK6 (G0/G1 phase regulators) at 5 h, cyclin E1 (G1/S phase regulator) at 7 h and cyclin A and CDK1 (S phase regulators) at 9 h post-infusion compared to A group. In addition, the mRNA expression levels of apoptotic genes, i.e., caspase 3, caspase 9 and Bax at 5 h post-infusion were upregulated (P < 0.05) in group B compared to group A. The present study demonstrated that butyrate improved ruminal epithelial growth through concurrent and time-dependent changes in the expressions of genes involved in cell proliferation and apoptosis. It seems that the rate of proliferation was higher than the apoptosis which was reflected in epithelial growth.

RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton

The RhoA/ROCK signaling pathway regulates cell morphology, adhesion, proliferation, and migration. In this study, we investigated the regulatory role of RhoA/ROCK signaling on PDGF-BB-mediated smooth muscle phenotypic modulation and vascular remodeling and clarified the molecular mechanisms behind these effects. PDGF-BB treatment induced the activation of RhoA, ROCK, PDGF-Rβ, and the expression of PDGF-Rβ in HA-VSMCs (human aortic vascular smooth muscle cells). PDGF-Rβ inhibition and RhoA suppression blocked PDGF-BB-induced RhoA activation and ROCK induction. In addition, PDGF-BB-mediated cell proliferation and migration were suppressed by PDGF-Rβ inhibition, RhoA suppression, and ROCK inhibition, suggesting that PDGF-BB promotes phenotypic modulation of HA-VSMCs by activating the RhoA/ROCK pathway via the PDGF receptor. Moreover, suppressing both ROCK1 and ROCK2 blocked cell cycle progression from G0/G1 to S phase by decreasing the transcription and protein expression of cyclin D1, CDK2, and CDK4 via JNK/c-Jun pathway, thus reducing cell proliferation in PDGF-BB-treated HA-VSMCs. ROCK1 deletion, rather than ROCK2 suppression, significantly inhibited PDGF-BB-induced migration by reducing the expression of vimentin and preventing the remodeling of vimentin and phospho-vimentin. Furthermore, ROCK1 deletion suppressed vimentin by inhibiting the phosphorylation of Smad2/3 and the nuclear translocation of Smad4. These findings suggested that ROCK1 and ROCK2 might play different roles in PDGF-BB-mediated cell proliferation and migration in HA-VSMCs. In addition, PDGF-BB and its receptor participated in neointima formation and vascular remodeling by promoting cell cycle protein expression via the JNK pathway and enhancing vimentin expression in a rat balloon injury model; effects that were inhibited by treatment with fasudil. Together, the results of this study reveal a novel mechanism through which RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.

Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest.

Diosmetin, a plant flavonoid, has been shown to exert promising effects on prostate cancer cells as an anti-proliferative and anticancer agent. In this study, using western blot analysis for protein expression and flow cytometry for cell cycle analysis, we determined that the treatment of the LNCaP and PC-3 prostate cancer cells with diosmetin resulted in a marked decrease in cyclin D1, Cdk2 and Cdk4 expression levels (these proteins remain active in the G0-G1 phases of the cell cycle). These changes were accompanied by a decrease in c-Myc and Bcl-2 expression, and by an increase in Bax, p27Kip1 and FOXO3a protein expression, which suggests the potential modulatory effects of diosmetin on protein transcription. The treatment of prostate cancer cells with diosmetin set in motion an apoptotic machinery by inhibiting X-linked inhibitor of apoptosis (XIAP) and increasing cleaved PARP and cleaved caspase-3 expression levels. On the whole, the findings of this study provide an in-depth analysis of the molecular mechanisms responsible for the regulatory effects of diosmetin on key molecules that perturb the cell cycle to inhibit cell growth, and suggest that diosmetin may prove to be an effective anticancer agent for use in the treatment of prostate cancer in the future.

Molecular Mechanisms of Apoptosis in HepaRG Cell Line Induced by Polyphyllin VI via the Fas Death Pathway and Mitochondrial-Dependent Pathway.

Polyphyllin VI, which is an active saponin, is mainly isolated from traditional medicinal plant Paris polyphylla, which causes liver damage in rats. In the present study, we aimed to explore the potential cytotoxicity of polyphyllin VI on the growth of HepaRG cells and to determine the molecular mechanism. The results revealed that polyphyllin VI changed cell morphology and induced apoptosis in HepaRG cells. Flow cytometric assay displayed that polyphyllin VI promoted the generation of reactive oxygen species (ROS), depolarized the mitochondrial membrane potential (MMP), and induced S phase cell cycle arrest by decreasing the expression of cyclin A2 and CDK2, while significantly increasing the expression of p21 protein. Polyphyllin VI induced the release of cytochrome c from the mitochondria to the cytosol and activated Fas, caspase-3, -8, -9, and PARP proteins. Pretreatment with NAC and Z-VAD-FMK (ROS scavenger and caspase inhibitor, respectively) on HepaRG cells increased the percentage of viable cells, which indicated that polyphyllin VI induced cell apoptosis through mitochondrial pathway by the generation of ROS and Fas death-dependent pathway. All of the effects are in dose- and time-dependent manners. Taken together, these findings emphasize the necessity of risk assessment to polyphyllin VI and offer an insight into polyphyllin VI-induced apoptosis of HepaRG cells.

Inhibition of MALAT1 sensitizes liver cancer cells to 5-flurouracil by regulating apoptosis through IKKα/NF-κB pathway

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in tumor cell growth process. However, its role and molecular mechanism in liver cancer is still not fully understood. In this study, we found that MALAT1 was significantly expressed in liver cancer cell lines. And knockdown of MALAT1 suppressed proliferation, migration and invasion of HepG2 cells, accompanied with decrease of Rho-associated coiled-coil-forming protein kinase 1 (ROCK1), α-smooth muscle actin (α-SMA), N-cadherin, Vimentin and TWIST. Significantly, MALAT1 deletion sensitized HepG2 cells to 5-FU-induced cell cycle arrest in G1 phase, as evidenced by the significant reduction in Cyclin D1 and CDK4 and increase in p53, p21 and p27 protein levels. In addition, MALAT1 knockdown triggered 5-FU induced apoptosis in HepG2 cells by inducing intrinsic apoptosis-related signals, including Cyto-c, Apaf-1, cleaved Caspase-9/-7/-3 and poly (ADP-ribose) polymerase (PARP). Furthermore, phosphorylated nuclear factor-κB (p-NF-κB) was also down-regulated by MALAT1 silence. Importantly, suppression of IKKα/NF-κB significantly elevated apoptosis and reduced liver cancer cell viability in MALAT1-knockdown cells with 5-FU incubation. The nude mice transplantation model also confirmed the promoted sensitivity of MALAT1-silenced HepG2 cells to 5-FU by blocking tumor cell proliferation and inducing apoptosis. Therefore, our data supplied a potential mechanism by which knockdown of MALAT1 might play an important role in augmenting sensitivity of HepG2 cells to 5-FU in therapeutic approaches, demonstrating suppressing of MALAT1 may serve as a combination with chemotherapeutic agents in liver cancer treatment.

Octyl gallate and gallic acid isolated from Terminalia bellarica regulates normal cell cycle in human breast cancer cell lines

Herbal medicines stand unique and effective in treating human diseases. Terminalia bellarica (T. bellarica) is a potent medicinal herb, with a wide range of pharmacological activities. The present study was aimed to evaluate the effect of octyl gallate (OG) and gallic acid (GA) isolated from methanolic fruit extract of T. bellirica to inhibit the survival of breast cancer cells (MCF-7 & MDA-MB-231). Both OG & GA exhibited decreased MCF-7 & MDA-MB-231 survival and induced apoptosis, with IC50 value of OG and GA as 40 μM and 80 μM respectively. No toxic effect was observed on normal breast cells (MCF-10A). The compounds inhibited cell cycle progression by altering the expression of the cell cycle regulators (Cyclin D1, D3, CDK-4, CDK-6, p18 INK4, p21Waf-1 and p27 KIP). Octyl gallate was more effective at low concentrations than GA. In-silico results provided stable interactions between the compounds and target proteins. The present investigation proved the downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators inducing apoptosis in compound-treated breast cancer cells. Hence, both the compounds may serve as potential anticancer agents and could be developed as breast cancer drugs, with further explorations.

Hypoxia-induced autophagy activation through NF-κB pathway regulates cell proliferation and migration to induce pulmonary vascular remodeling

ObjectivesThis study aimed to investigate whether autophagy mediated cell proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia and to validate the underlying mechanism. MethodsThe rat model of chronic hypoxia-induced pulmonary vascular remodeling was established. Rat primary PASMCs were isolated and cultured in vitro in hypoxia or normoxia to explore the underlying mechanism. ResultsHypoxia exposure for 21 days induced pulmonary vascular remodeling in rats, accompanied by enhanced proliferation and decreased apoptosis, as well as induced expression of autophagic marker LC3II in their lungs. Furthermore, in vitro assays revealed that hypoxia exposure significantly promoted cell proliferation and migration, but inhibited apoptosis of PASMCs compared with normoxia treatment. Besides, hypoxia exposure led to an increase of cell fractions in S phase and a decrease in G0/G1 of PASMCs, whereas had no significant effect on cell population in G2/M phase. Moreover, the autophagy inhibitor 3-MA significantly decreased the hypoxia-induced upregulated expression of LC3II in PASMCs and abrogated the effect of hypoxia on cell proliferation, cell apoptosis, cell cycle, and cell migration of PASMCs. Additionally, inhibition of NF-κB pathway by PDTC suppressed the hypoxia-induced upregulation of NF-κB activity, LC3II, and cell cycle regulators (cyclin D1, CDK4, and CDK6) in PASMCs, and abolished the hypoxia-mediated regulation of cell proliferation, apoptosis, cycle, and migration of PASMCs. ConclusionIn summary, hypoxia could induce autophagy activation through NF-κB pathway, and thereby regulate cell proliferation and migration to induce pulmonary vascular remodeling.

BRD7 inhibits the Warburg effect and tumor progression through inactivation of HIF1\u03b1/LDHA axis in breast cancer

The bromodomain-containing protein 7 (BRD7) was first identified as a tumor suppressor in nasopharyngeal carcinoma and has critical roles in cancer development and progression. However, the regulatory roles and mechanisms of BRD7 in cancer metabolism are still unknown. In this study, we demonstrated that BRD7 was lowly expressed in breast cancer tissues and was identified as a poor prognostic factor in breast cancer. Meanwhile, BRD7 could suppress cell proliferation, initiate cell apoptosis and reduce aerobic glycolysis, suggesting that BRD7 plays a tumor suppressive roles in breast cancer. Mechanistically, BRD7 could negatively regulate a critical glycolytic enzyme LDHA through directly interaction with its upstream transcription factor, HIF1α, facilitating degradation of HIF1α mediated by ubiquitin–proteasome pathway. Moreover, restoring the expression of LDHA in breast cancer cells could reverse the effect of BRD7 on aerobic glycolysis, cell proliferation, and tumor formation, as well as the expression of cell cycle and apopotosis related molecules such as cyclin D1, CDK4, P21, and c-PARP both in vitro and in vivo. Taken together, these results indicate that BRD7 acts as a tumor suppressor in breast cancer and represses the glycolysis and tumor progression through inactivation of HIF1α/LDHA transcription axis.

Knockdown of NUP160 inhibits cell proliferation, induces apoptosis, autophagy and cell migration, and alters the expression and localization of podocyte associated molecules in mouse podocytes

Genetic mutations in dozens of monogenic genes can lead to serious podocyte dysfunction, which is a major cause of steroid-resistant nephrotic syndrome (SRNS). The NUP160 gene is expressed in both human kidney and mouse kidney. However, whether knockdown of NUP160 impairs podocytes has not yet been established. Therefore, we knocked down NUP160 by targeted short hairpin RNA (shRNA) in conditionally immortalized mouse podocytes and observed the effect of NUP160 knockdown on the proliferation, apoptosis, autophagy and cell migration of podocytes. We also investigated the effect of NUP160 knockdown on the expression and localization of podocyte associated molecules, such as nephrin, podocin, CD2AP and α-actinin-4. The knockdown of NUP160 significantly inhibited the proliferation of podocytes by decreasing the expression of both cyclin D1 and CDK4, increasing the expression of p27, and inducing S phase arrest. The knockdown of NUP160 promoted the apoptosis and autophagy of podocytes, and enhanced cell migration. The knockdown of NUP160 decreased the expression of nephrin, podocin and CD2AP in podocytes, and increased the expression of α-actinin-4. The knockdown of NUP160 also altered the subcellular localization of nephrin, podocin and CD2AP in podocytes. These results suggest that the knockdown of NUP160 impairs mouse podocytes, i.e. inhibiting cell proliferation, inducing apoptosis, autophagy and cell migration of mouse podocytes, and altering the expression and localization of podocyte associated molecules, including nephrin, podocin, CD2AP and α-actinin-4.

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms

Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

Morphological adaptation of sheep\u2019s rumen epithelium to high-grain diet entails alteration in the expression of genes involved in cell cycle regulation, cell proliferation and apoptosis

BackgroundThe objectives of this study were to characterize changes in the relative mRNA expression of candidate genes and proteins involved in cell cycle regulation, cell proliferation and apoptosis in the ruminal epithelium (RE) of sheep during high-grain (HG) diet adaptation.ResultsTwenty sheep were assigned to four groups with five animals each. These animals were assigned to different periods of HG diet (containing 40% forage and 60% concentrate mix) feeding. The HG groups received an HG diet for 7 (G7, n = 5), 14 (G14, n = 5) and 28 d (G28, n = 5), respectively. In contrast, the control group (CON, n = 5) was fed the forage-based diet for 28 d. The results showed that HG feeding linearly decreased (P < 0.001) the ruminal pH, and increased the concentrations of ruminal total volatile fatty acid (linear, P = 0.001), butyrate (linear, P < 0.001), valerate (quadratic P = 0.029) and the level of IGF-1 (quadratic, P = 0.043) in plasma. The length (quadratic, P = 0.004), width (cubic, P = 0.015) and surface of the ruminal papillae (linear, P = 0.003) were all enlarged after 14 d of HG diet feeding. HG feeding cubically increased the number of cell layers forming the stratum corneum (SC, P < 0.001) and the thickness of the SC (P < 0.001) and stratum basale (P < 0.001). The proportion of basal layer cells in the RE decreased (linear, P < 0.001) in the G0/G1-phase, but it increased linearly (P = 0.006) in the S-phase and cubically (P = 0.004) in the G2/M-phases. The proportion of apoptosis cells in G7, G14 and G28 was reduced compared to the CON (quadratic, P < 0.001). HG diet feeding linearly decreased the mRNA expression of Cyclin E1 (P = 0.021) and CDK-2 (P = 0.001) and (P = 0.027) the protein expression of Cyclin E1. Feeding an HG diet linearly increased the mRNA expression of genes IGFBP-2 (P = 0.034) and IGFBP 5 (P < 0.009), while linearly decreasing (P < 0.001) the IGFBP 3 expression. The expression of cell apoptosis gene Caspase 8 decreased (quadratic, P = 0.012), while Bad mRNA expression tended to decrease (cubic, P = 0.053) after HG feeding.ConclusionsThese results demonstrated sequential changes in rumen papillae size, cell cycle regulation and the genes involved in proliferation and apoptosis as time elapsed in feeding a high-grain diet to sheep.

Menage a Trois 1-Mediated p53 Ubiqutin Degradation Promotes Colorectal Cancer Malignance

Background MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, high expresses in various cancers and is involved in cancer pathogenesis. However, mechanisms underlying its regulation in carcinogenesis are unclear. Methods Colorectal cancer (CRC) tissue microarray was used to evaluate MNAT1 expressions using immunohistchemistry, MNAT1 in CRC cell lines was detected using Western-blotting. Growths of the cells tansfected with MNAT1 or shMNAT1 were observed using MTT and colony formation. Affects of MNAT1 on p53/TP53 expressions were analyzed using Western-blotting and Real-time PCR. Immunoprecipitation assay was used to analyze interaction p53 with MNAT1, p53 downstream molecules were detected. The apoptosis of CRC cells with MNAT1 or shMNAT1 was analyzed using flow cytometry. BABL/c nude mice were used to observe effects of MNAT1 on CRC cell growth in vivo. Findings MNAT1 was highly expressed in CRC tissues and cells, and MNAT1 expressions were associated with CRC carcinogenesis and poor patient outcomes. MNAT1-knockin increased CRC cell growth and colony formation, and MNAT1-knockdown decreased cell growth, motility and invasion. MNAT1 physically interacted with p53, MNAT1 also increased interaction of MDM2 with p53. Strikingly, MNAT1 mediates p53 ubiquitin-degradation. MNAT1 shortened p53 half-life. Moreover, MNAT1 induced RAD51 and reduced PARP and BAX expression through p53, and inhibited CRC cell apoptosis. MANT1 promoted tumor growths in nude mice following decreased p53. Interpretation MNAT1 binds to p53, mediates p53 ubiquitin-degradation through MDM2, increases CRC cell growth and decreases apoptosis, and finally promotes CRC malignance. Funding: National Natural Science Foundation of China (81372282, 81402265, 81502346). Declaration of Interest: None. Ethical Approval: All procedures were consistent with the National Institutes of Health Guide and approved by institutional board with patients’ written consent. This study was evaluated and approved by the Ethics Committee of the Affiliated Cancer Hospital of Xiangya Medical School, Central South University. All animal experiments were performed according to the National Institutes of Health Animal Use Guidelines on the Use of Experimental Animals.