Lithium Causes G2 Arrest of Renal Principal Cells

PubMed Central

de Groot, Theun; Alsady, Mohammad; Jaklofsky, Marcel; Otte-Höller, Irene; Baumgarten, Ruben; Giles, Rachel H.

2014-01-01

Vasopressin-regulated expression and insertion of aquaporin-2 channels in the luminal membrane of renal principal cells is essential for urine concentration. Lithium affects urine concentrating ability, and approximately 20% of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder characterized by polyuria and polydipsia. Lithium-induced NDI is caused by aquaporin-2 downregulation and a reduced ratio of principal/intercalated cells, yet lithium induces principal cell proliferation. Here, we studied how lithium-induced principal cell proliferation can lead to a reduced ratio of principal/intercalated cells using two-dimensional and three-dimensional polarized cultures of mouse renal collecting duct cells and mice treated with clinically relevant lithium concentrations. DNA image cytometry and immunoblotting revealed that lithium initiated proliferation of mouse renal collecting duct cells but also increased the G2/S ratio, indicating G2/M phase arrest. In mice, treatment with lithium for 4, 7, 10, or 13 days led to features of NDI and an increase in the number of principal cells expressing PCNA in the papilla. Remarkably, 30%–40% of the PCNA-positive principal cells also expressed pHistone-H3, a late G2/M phase marker detected in approximately 20% of cells during undisturbed proliferation. Our data reveal that lithium treatment initiates proliferation of renal principal cells but that a significant percentage of these cells are arrested in the late G2 phase, which explains the reduced principal/intercalated cell ratio and may identify the molecular pathway underlying the development of lithium-induced renal fibrosis. PMID:24408872

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

PubMed

Wang, Juan; Huang, Shujuan; Xing, Lingxiao; Cui, Jinfeng; Tian, Ziqiang; Shen, Haitao; Jiang, Xiujuan; Yan, Xia; Wang, Junling; Zhang, Xianghong

2015-11-01

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest

Influence of environmental pH on G2-phase arrest caused by ionizing radiation.

PubMed

Park, Heon Joo; Lee, Sang Hwa; Chung, HyunSook; Rhee, Yun Hee; Lim, Byung Uk; Ha, Sung Whan; Griffin, Robert J; Lee, Hyung Sik; Song, Chang Won; Choi, Eun Kyung

2003-01-01

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.

Robust G2 pausing of adult stem cells in Hydra.

PubMed

Buzgariu, Wanda; Crescenzi, Marco; Galliot, Brigitte

2014-01-01

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

PubMed

Stuart, David T

2017-01-01

Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

β2-adrenoceptor blockage induces G1/S phase arrest and apoptosis in pancreatic cancer cells via Ras/Akt/NFκB pathway.

PubMed

Zhang, Dong; Ma, Qingyong; Wang, Zheng; Zhang, Min; Guo, Kun; Wang, Fengfei; Wu, Erxi

2011-11-26

Smoking and stress, pancreatic cancer (PanCa) risk factors, stimulate nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and catecholamines production respectively. NNK and catecholamine bind the β-adrenoceptors and induce PanCa cell proliferation; and we have previously suggested that β-adrenergic antagonists may suppress proliferation and invasion and stimulate apoptosis in PanCa. To clarify the mechanism of apoptosis induced by β2-adrenergic antagonist, we hypothesize that blockage of the β2-adrenoceptor could induce G1/S phase arrest and apoptosis and Ras may be a key player in PanCa cells. The β1 and β2-adrenoceptor proteins were detected on the cell surface of PanCa cells from pancreatic carcinoma specimen samples by immunohistochemistry. The β2-adrenergic antagonist ICI118,551 significantly induced G1/S phase arrest and apoptosis compared with the β1-adrenergic antagonist metoprolol, which was determined by the flow cytometry assay. β2-adrenergic antagonist therapy significantly suppressed the expression of extracellular signal-regulated kinase, Akt, Bcl-2, cyclin D1, and cyclin E and induced the activation of caspase-3, caspase-9 and Bax by Western blotting. Additionally, the β2-adrenergic antagonist reduced the activation of NFκB in vitro cultured PanCa cells. The blockage of β2-adrenoceptor markedly induced PanCa cells to arrest at G1/S phase and consequently resulted in cell death, which is possibly due to that the blockage of β2-adrenoceptor inhibited NFκB, extracellular signal-regulated kinase, and Akt pathways. Therefore, their upstream molecule Ras may be a key factor in the β2-adrenoceptor antagonist induced G1/S phase arrest and apoptosis in PanCa cells. The new pathway discovered in this study may provide an effective therapeutic strategy for PanCa.

ABT-263 induces G1/G0-phase arrest, apoptosis and autophagy in human esophageal cancer cells in vitro.

PubMed

Lin, Qing-Huan; Que, Fu-Chang; Gu, Chun-Ping; Zhong, De-Sheng; Zhou, Dan; Kong, Yi; Yu, Le; Liu, Shu-Wen

2017-12-01

Both the anti- and pro-apoptotic members of the Bcl-2 family are regulated by a conserved Bcl-2 homology (BH3) domain. ABT-263 (Navitoclax), a novel BH3 mimetic and orally bioavailable Bcl-2 family inhibitor with high affinity for Bcl-xL, Bcl-2 and Bcl-w has entered clinical trials for cancer treatment. But the anticancer mechanisms of ABT-263 have not been fully elucidated. In this study we investigated the effects of ABT-263 on human esophageal cancer cells in vitro and to explore its anticancer mechanisms. Treatment with ABT-263 dose-dependently suppressed the viability of 3 human esophageal cancer cells with IC 50 values of 10.7±1.4, 7.1±1.5 and 8.2±1.6 μmol/L, in EC109, HKESC-2 and CaES-17 cells, respectively. ABT-263 (5-20 μmol/L) dose-dependently induced G 1 /G 0 -phase arrest in the 3 cancer cell lines and induced apoptosis evidenced by increased the Annexin V-positive cell population and elevated levels of cleaved caspase 3, cleaved caspase 9 and PARP. We further demonstrated that ABT-263 treatment markedly increased the expression of p21 Waf1/Cip1 and decreased the expression of cyclin D1 and phospho-Rb (retinoblastoma tumor suppressor protein) (Ser780) proteins that contributed to the G 1 /G 0 -phase arrest. Knockdown of p21 Waf1/Cip1 attenuated ABT-263-induced G 1 /G 0 -phase arrest. Moreover, ABT-263 treatment enhanced pro-survival autophagy, shown as the increased LC3-II levels and decreased p62 levels, which counteracted its anticancer activity. Our results suggest that ABT-263 exerts cytostatic and cytotoxic effects on human esophageal cancer cells in vitro and enhances pro-survival autophagy, which counteracts its anticancer activity.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

PubMed

Minemoto, Yuzuru; Uchida, Sanae; Ohtsubo, Motoaki; Shimura, Mari; Sasagawa, Toshiyuki; Hirata, Masato; Nakagama, Hitoshi; Ishizaka, Yukihito; Yamashita, Katsumi

2003-04-01

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

Requirement of ClC-3 in G0/G1 to S Phase Transition Induced by IGF-1 via ERK1/2-Cyclins Cascade in Multiple Myeloma Cells.

PubMed

Du, Yu; Tu, Yong-Sheng; Tang, Yong-Bo; Huang, Yun-Ying; Zhou, Fang-Min; Tian, Tian; Li, Xiao-Yan

2018-06-01

ClC-3 is involved in the proliferation and migration of several cancer cells. However, ClC-3 expression and its role of cell-cycle control in multiple myeloma (MM) has not yet been investigated. MM cells were treated with different concentrations of IGF (30, 100, 300 ng/mL), and their proliferation was examined by CCK-8. The effects of ClC-3 on cell cycle progression was detected by flow cytometry. Western blot was used to analyze the relative levels of ClC3, CD138, P21, P27, CDK, p-Erk1/2, and t-Erk1/2 protein expression. Transfection of RPMI8226 with gpClC-3 cDNA and siRNA alters the expression of ClC-3. We compared the expression of ClC-3 in primary myeloma cells and in MM cell lines (U266 and RPMI8266) with that in normal plasma cells (PCs) from normal subjects and found that myeloma cells from patients and MM cell lines had significantly higher expression of ClC-3. Additionally, silencing of ClC-3 with the small interfering RNA (siRNA) that targets human ClC-3 decreased proliferation of RPMI8226 after IGF-1 treatment and slowed cell cycle progression from G0/G1 to S phase, which was associated with diminished phosphorylation of ERK1/2, down-expression of cyclin E, cyclin D1 and up-regulation of p27 and p21. By contrast, overexpression of ClC-3 potentiated cell proliferation induced by IGF-1, raised the percentage of S phase cells, enhanced phosphorylation of ERK1/2, downregulated p27 and p21 and upregulated cyclin E and cyclin D1. ClC-3 accelerated G0/G1 to S phase transition in the cell cycle by modulating ERK1/2 kinase activity and expression of G1/S transition related proteins, making ClC-3 an attractive therapeutic target in MM.

Cytotoxic effects of 2-methoxyestradiol in the hepatocellular carcinoma cell line HepG2.

PubMed

El Naga, Reem N Abou; El-Demerdash, Ebtehal; Youssef, Samar S; Abdel-Naim, Ashraf B; El-Merzabani, Mahmoud

2009-01-01

The study was designed to examine the potential cytotoxicity of 2-methoxyestradiol (2ME2), a natural 17beta-estradiol metabolite, in hepatocellular carcinoma and the possible underlying mechanisms for this cytotoxicity. The cell line HepG2 was treated with different concentrations of 2ME2 for 48 and 72 h. Using the sulforhodamine B assay, HepG2 was sensitive to the cytotoxic effect of 2ME2. 2ME2 induced cell arrest at the G(2)/M phase and a significant high percentage of apoptotic cells compared to the control group. Also, 2ME2 induced a significant increase in caspase 9 enzymatic activity after 48 and 72 h of treatment compared with control values. The DNA laddering was observed only in cells treated for 72 h. Furthermore, 2ME2 induced a significant decrease in the expression levels of vascular endothelial growth factor (VEGF) gene compared to the control values. 2ME2 exerts cytotoxic activity in the HepG2 cell line by preferential cell blocking at the G(2)/M phase as well as induction of apoptosis as evidenced by increased caspase 9 enzymatic activity and observed DNA laddering in 2ME2-treated HepG2 cells. In addition, a reduction in hypervascularity is an important postulated mechanism as indicated by the significant reduction in the expression of VGEF, one of the most important angiogenic factors.

Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases.

PubMed

Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M

2017-01-01

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G 1 -phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G 1 -phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21 WAF1/CIP1 , a negative regulator of the G 1 phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G 1 -phase cyclins and CDKs, and upregulating p21 WAF1/CIP1 , which leads to G 1 -phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin

2014-08-08

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562more » cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.« less

Examination of a modified cell cycle synchronization method and bovine nuclear transfer using synchronized early G1 phase fibroblast cells.

PubMed

Urakawa, Manami; Ideta, Atsushi; Sawada, Tokihiko; Aoyagi, Yoshito

2004-08-01

Somatic cell nuclear transfer has a low success rate, due to a high incidence of fetal loss and increased perinatal morbidity/mortality. One factor that may affect the successful development of nuclear transfer embryos is the cell cycle stage of the donor cell. In order to establish a cell cycle synchronization method that can consistently produce cloned embryos and offspring, we examined the effects of different combinations of three cell treatments on the recovery rate of mitotic phase cells using bovine fetal fibroblasts. In the first experiment, we examined the recovery rate of mitotic phase cells by a combination of treatment with a metaphase arrestant (1 microM 2-methoxyestradiol), shaking the plate and selecting cells with a diameter of 20 microns. As a result, 99% of mitotic phase cells were recovered by repeating the combined treatment of metaphase arrestant and shaking, and collection of cells with a specific diameter. In the second experiment, nuclear transfer was carried out using early G1 phase cells by choosing pairs of bridged cells derived from mitotic phase cells recovered by the combined treatment of 1 microM 2-methoxyestradiol and shaking, and collection of cells with a diameter of 20 microns. The reconstructed embryos were transferred to recipient heifers to determine post-implantation development. Development of embryos reconstructed from early G1 phase cells from the >/=6 cells stage on Day 3 to the morula-blastocyst stage on Day 6 was 100%. Ten blastocysts constructed from two cell lines were transferred into 10 recipient heifers. Nine of the 10 recipients delivered single live calves. In conclusion, mitotic phase bovine fibroblast cells were easily recovered by the combined treatments of 1 microM 2-methoxyestradiol, shaking, and selecting cells of the appropriate diameter. Furthermore, nuclear transfer using cells in the early G1 phase as donor cells gave a high rate of offspring production.

Anti-hepatocarcinoma effects of resveratrol nanoethosomes against human HepG2 cells

NASA Astrophysics Data System (ADS)

Meng, Xiang-Ping; Zhang, Zhen; Chen, Tong-sheng; Wang, Yi-fei; Wang, Zhi-ping

2017-02-01

Hepatocarcinoma, a malignant cancer, threaten human life badly. It is a current issue to seek the effective natural remedy from plant to treat cancer due to the resistance of the advanced hepatocarcinoma to chemotherapy. Resveratrol (Res) has been widely investigated with its strong anti-tumor activity. However, its low oral bioavailability restricts its wide application. In this study, we prepared resveratrol nanoethosomes (ResN) via ethanol injection method. The in vitro anti-hepatocarcinoma effects of ResN relative to efficacy of bulk Res were evaluated on proliferation and apoptosis of human HepG2 cells. ResN were spherical vesicles and its particle diameter, zeta potential were (115.8 +/- 1.3) nm and (-12.8 +/- 1.9) mV, respectively. ResN exhibited significant inhibitory effects against human HepG2 cells by MTT assay, and the IC50 value was 49.2 μg/ml (105.4 μg/ml of Res bulk solution). By flow cytometry assay, there was an increase in G2/M phase cells treated with ResN. The results demonstrated ResN could effectively block the G2/M phase of HepG2 cells, which can also enhance the inhibitory effect of Res against HepG2 cells.

Glycyrrhetinic acid induces G1-phase cell cycle arrest in human non-small cell lung cancer cells through endoplasmic reticulum stress pathway

PubMed Central

ZHU, JIE; CHEN, MEIJUAN; CHEN, NING; MA, AIZHEN; ZHU, CHUNYAN; ZHAO, RUOLIN; JIANG, MIAO; ZHOU, JING; YE, LIHONG; FU, HAIAN; ZHANG, XU

2015-01-01

Glycyrrhetinic acid (GA) is a natural compound extracted from liquorice, which is often used in traditional Chinese medicine. The purpose of the present study was to investigate the antitumor effect of GA in human non-small cell lung cancer (NSCLC), and its underlying mechanisms in vitro. We have shown that GA suppressed the proliferation of A549 and NCI-H460 cells. Flow cytometric analysis showed that GA arrested cell cycle in G0/G1 phase without inducing apoptosis. Western blot analysis indicated that GA mediated G1-phase cell cycle arrest by upregulation of cyclin-dependent kinase inhibitors (CKIs) (p18, p16, p27 and p21) and inhibition of cyclins (cyclin-D1, -D3 and -E) and cyclin-dependent kinases (CDKs) (CDK4, 6 and 2). GA also maintained pRb phosphorylation status, and inhibited E2F transcription factor 1 (E2F-1) in both cell lines. GA upregulated the unfolded proteins, Bip, PERK and ERP72. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggered the unfolded protein response (UPR), which could be the mechanism by which GA inhibited cell proliferation in NSCLC cells. GA then coordinated the induction of ER chaperones, which decreased protein synthesis and induced cell cycle arrest in the G1 phase. This study provides experimental evidence to support the development of GA as a chemotherapeutic agent for NSCLC. PMID:25573651

Enterolactone induces G1-phase cell cycle arrest in non-small cell lung cancer cells by down-regulating cyclins and cyclin-dependent kinases

PubMed Central

Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M.

2017-01-01

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG) which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anti-cancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study we investigated the anti-cancer effects of EL for several non-small cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The anti-proliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL- decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21WAF1/CIP1, a negative regulator of the G1-phase. The results suggest that EL inhibits the growth of NSCLC cell lines by down-regulating G1-phase cyclins and CDKs, and up-regulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy. PMID:28323486

H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia.

PubMed

Rybaczek, Dorota; Bodys, Aleksandra; Maszewski, Janusz

2007-09-01

Immunocytochemistry using alpha-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases alpha and delta, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX aggregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.

A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells.

PubMed

Hegde, Mahesh; Vartak, Supriya V; Kavitha, Chandagirikoppal V; Ananda, Hanumappa; Prasanna, Doddakunche S; Gopalakrishnan, Vidya; Choudhary, Bibha; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2017-05-31

Chemically synthesized small molecules play important role in anticancer therapy. Several chemical compounds have been reported to damage the DNA, either directly or indirectly slowing down the cancer cell progression by causing a cell cycle arrest. Direct or indirect reactive oxygen species formation causes DNA damage leading to cell cycle arrest and subsequent cell death. Therefore, identification of chemically synthesized compounds with anticancer potential is important. Here we investigate the effect of benzothiazole derivative (5g) for its ability to inhibit cell proliferation in different cancer models. Interestingly, 5g interfered with cell proliferation in both, cell lines and tumor cells leading to significant G2/M arrest. 5g treatment resulted in elevated levels of ROS and subsequently, DNA double-strand breaks (DSBs) explaining observed G2/M arrest. Consistently, we observed deregulation of many cell cycle associated proteins such as CDK1, BCL2 and their phosphorylated form, CyclinB1, CDC25c etc. Besides, 5g treatment led to decreased levels of mitochondrial membrane potential and activation of apoptosis. Interestingly, 5g administration inhibited tumor growth in mice without significant side effects. Thus, our study identifies 5g as a potent biochemical inhibitor to induce G2/M phase arrest of the cell cycle, and demonstrates its anticancer properties both ex vivo and in vivo.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

PubMed

Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Gravitational force modulates G2/M phase exit in mechanically unloaded myoblasts

PubMed Central

Benavides Damm, Tatiana; Franco-Obregón, Alfredo; Egli, Marcel

2013-01-01

Prolonged spaceflight gives rise to muscle loss and reduced strength, a condition commonly referred to as space atrophy. During exposure to microgravity, skeletal muscle myoblasts are mechanically unloaded and respond with attenuated cell proliferation, slowed cell cycle progression, and modified protein expression. To elucidate the underlying mechanisms by which muscle mass declines in response to prolonged microgravity exposure, we grew C2C12 mouse muscle cells under conditions of simulated microgravity (SM) and analyzed their proliferative capacity, cell cycle progression, and cyclin B and D expression. We demonstrated that the retarded cell growth observed in SM was correlated with an approximate 16 h delay in G2/M phase progression, where cells accumulated specifically between the G2 checkpoint and the onset of anaphase, concomitantly with a positive expression for cyclin B. The effect was specific for gravitational mechanical unloading as cells grown under conditions of hypergravity (HG, 4 g) for similar durations of time exhibited normal proliferation and normal cell cycle progression. Our results show that SM and HG exert phenomenological distinct responses over cell cycle progression. The deficits of SM can be restored by terrestrial gravitational force, whereas the effects of HG are indistinguishable from the 1 g control. This suggests that the mechanotransduction apparatus of cells responds differently to mechanical unloading and loading. PMID:23974110

Rejoining of isochromatid breaks induced by heavy ions in G2-phase normal human fibroblasts

NASA Technical Reports Server (NTRS)

Kawata, T.; Durante, M.; Furusawa, Y.; George, K.; Ito, H.; Wu, H.; Cucinotta, F. A.

2001-01-01

We reported previously that exposure of normal human fibroblasts in G2 phase of the cell cycle to high-LET radiation produces a much higher frequency of isochromatid breaks than exposure to gamma rays. We concluded that an increase in the production of isochromatid breaks is a signature of initial high-LET radiation-induced G2-phase damage. In this paper, we report the repair kinetics of isochromatid breaks induced by high-LET radiation in normal G2-phase human fibroblasts. Exponentially growing human fibroblasts (AG1522) were irradiated with gamma rays or energetic carbon (290 MeV/nucleon), silicon (490 MeV/nucleon), or iron (200 MeV/nucleon) ions. Prematurely condensed chromosomes were induced by calyculin A after different postirradiation incubation times ranging from 0 to 600 min. Chromosomes were stained with Giemsa, and aberrations were scored in cells at G2 phase. G2-phase fragments, the result of the induction of isochromatid breaks, decreased quickly with incubation time. The curve for the kinetics of the rejoining of chromatid-type breaks showed a slight upward curvature with time after exposure to 440 keV/microm iron particles, probably due to isochromatid-isochromatid break rejoining. The formation of chromatid exchanges after exposure to high-LET radiation therefore appears to be underestimated, because isochromatid-isochromatid exchanges cannot be detected. Increased induction of isochromatid breaks and rejoining of isochromatid breaks affect the overall kinetics of chromatid-type break rejoining after exposure to high-LET radiation.

Tubeimoside-1 induces oxidative stress-mediated apoptosis and G0/G1 phase arrest in human prostate carcinoma cells in vitro

PubMed Central

Yang, Jing-bo; Khan, Muhammad; He, Yang-yang; Yao, Min; Li, Yong-ming; Gao, Hong-wen; Ma, Tong-hui

2016-01-01

Aim: Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from the Chinese herbal medicine Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), has shown anticancer activities in various cancer cell lines. The aim of this study was to investigate the anticancer activity and molecular targets of TBMS1 in human prostate cancer cells in vitro. Methods: DU145 and P3 human prostate cancer cells were treated with TBMS1. Cell viability and apoptosis were detected. ROS generation, mitochondrial membrane potential and cell cycle profile were examined. Western blotting was used to measure the expression of relevant proteins in the cells. Results: TBMS1 (5–100 μmol/L) significantly suppressed the viability of DU145 and P3 cells with IC50 values of approximately 10 and 20 μmol/L, respectively. Furthermore, TBMS1 dose-dependently induced apoptosis and cell cycle arrest at G0/G1 phase in DU145 and P3 cells. In DU145 cells, TBMS1 induced mitochondrial apoptosis, evidenced by ROS generation, mitochondrial dysfunction, endoplasmic reticulum stress, modulated Bcl-2 family protein and cleaved caspase-3, and activated ASK-1 and its downstream targets p38 and JNK. The G0/G1 phase arrest was linked to increased expression of p53 and p21 and decreased expression of cyclin E and cdk2. Co-treatment with Z-VAD-FMK (pan-caspase inhibitor) could attenuate TBMS1-induced apoptosis but did not prevent G0/G1 arrest. Moreover, co-treatment with NAC (ROS scavenger), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) or salubrinal (ER stress inhibitor) significantly attenuated TBMS1-induced apoptosis. Conclusion: TBMS1 induces oxidative stress-mediated apoptosis in DU145 human prostate cancer cells in vitro via the mitochondrial pathway. PMID:27292614

Localized movement and morphology of UBF1-positive nucleolar regions are changed by γ-irradiation in G2 phase of the cell cycle

PubMed Central

Sorokin, Dmitry V; Stixová, Lenka; Sehnalová, Petra; Legartová, Soňa; Suchánková, Jana; Šimara, Pavel; Kozubek, Stanislav; Matula, Pavel; Skalníková, Magdalena; Raška, Ivan; Bártová, Eva

2015-01-01

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase. PMID:26208041

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

PubMed

Choi, Hyun Ju; Lim, Do Young; Park, Jung Han Yoon

2009-05-29

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

The O-methylated isoflavone, formononetin, inhibits human ovarian cancer cell proliferation by sub G0/G1 cell phase arrest through PI3K/AKT and ERK1/2 inactivation.

PubMed

Park, Sunwoo; Bazer, Fuller W; Lim, Whasun; Song, Gwonhwa

2018-05-15

Formononetin is an isoflavone that is extracted from red clovers or soy. It has anti-oxidant, anti-proliferative, and anti-tumor effects against cells in various diseases. Several cohort studies have indicated that phytoestrogen intake, including formononetin, could reduce the risk of various carcinogenesis. In fact, many case-control studies have indicated the potential value of flavonoids as drug supplements in the treatment of many cancer patients. However, the toxic effects and the anti-cancer mechanism of formononetin in ovarian cancer are unknown. We investigated the toxicological mechanism of formononetin in ES2 and OV90 ovarian cancer cells. Formononetin suppressed cell proliferation through sub G0/G1 phase arrest and increased apoptosis in both cell lines. Furthermore, it induced loss of mitochondrial membrane potential and generation of reactive oxygen species in ES2 and OV90 cells. The formononetin-mediated regulation of cell proliferation and apoptosis involved decreased phosphorylation of ERK1/2, P90RSK, AKT, P70S6K, and S6 proteins, and increased phosphorylation of P38 protein in ES2 and OV90 cells. Co-treatment of formononetin with pharmacological inhibitors (LY294002 or U0126) revealed additional anti-proliferative effects on the two human ovarian cancer cell types. Conclusively, the results indicate the potential value of formononetin as an anti-cancer agent in human ovarian cancer. © 2018 Wiley Periodicals, Inc.

Hyperglycemia and Anthocyanin Inhibit Quercetin Metabolism in HepG2 Cells.

PubMed

Hashimoto, Naoto; Blumberg, Jeffrey B; Chen, C-Y Oliver

2016-02-01

A high glucose (Glu) milieu promotes generation of reactive oxygen species, which may not only cause cellular damage, but also modulate phase II enzymes that are responsible for the metabolism of flavonoids. Thus, we examined the effect of a high Glu milieu on quercetin (Q) metabolism in HepG2 cells. HepG2 cells were grown for 3 days in Glu ranging from 5.5 to 50 mmol/L and/or cyanidin-3-glucoside (C3G) ranging from 0 to 25 μmol/L. Subsequently, the capacity of HepG2 cells to metabolize Q was assessed for up to 16 h. Q metabolites were analyzed by high-performance liquid chromatography. Four major Q metabolites were observed in the culture medium and inside the HepG2 cells. Three of these metabolites appear to be sulfated forms of Q or methylated Q, and one was a methylated Q. These metabolites and Q itself were reduced or tended to be reduced in cells grown in a high Glu compared to a normal Glu medium. Addition of C3G or superoxide dismutase plus catalase did not prevent or enhance reduction of Q metabolites. In vitro, a hyperglycemic milieu decreases the production of the principal Q metabolites in HepG2 cells, mediated through mechanisms independent of oxidative stress.

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

NASA Technical Reports Server (NTRS)

Wang, Xiang; Khadpe, Jay; Hu, Baocheng; Iliakis, George; Wang, Ya

2003-01-01

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Yong-Cheng; Su, Nan; Shi, Xiao-Jing

2015-01-15

Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore,more » Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.« less

Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes.

PubMed

Clare, Susan E; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z; Kim, J Julie; Khan, Seema A

2016-05-23

The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.

Murrayafoline A Induces a G0/G1-Phase Arrest in Platelet-Derived Growth Factor-Stimulated Vascular Smooth Muscle Cells

PubMed Central

Han, Joo-Hui; Kim, Yohan; Jung, Sang-Hyuk; Lee, Jung-Jin; Park, Hyun-Soo; Song, Gyu-Yong; Cuong, Nguyen Manh; Kim, Young Ho

2015-01-01

The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through G0/G1 to S phase of the cell cycle, as measured by [3H]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at G0/G1 phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis. PMID:26330754

Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

PubMed

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-04-12

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle.

PubMed

Yamane, Arito; Robbiani, Davide F; Resch, Wolfgang; Bothmer, Anne; Nakahashi, Hirotaka; Oliveira, Thiago; Rommel, Philipp C; Brown, Eric J; Nussenzweig, Andre; Nussenzweig, Michel C; Casellas, Rafael

2013-01-31

Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Effects on g2/m phase cell cycle distribution and aneuploidy formation of exposure to a 60 Hz electromagnetic field in combination with ionizing radiation or hydrogen peroxide in l132 nontumorigenic human lung epithelial cells.

PubMed

Jin, Hee; Yoon, Hye Eun; Lee, Jae-Seon; Kim, Jae-Kyung; Myung, Sung Ho; Lee, Yun-Sil

2015-03-01

The aim of the present study was to assess whether exposure to the combination of an extremely low frequency magnetic field (ELF-MF; 60 Hz, 1 mT or 2 mT) with a stress factor, such as ionizing radiation (IR) or H2O2, results in genomic instability in non-tumorigenic human lung epithelial L132 cells. To this end, the percentages of G2/M-arrested cells and aneuploid cells were examined. Exposure to 0.5 Gy IR or 0.05 mM H2O2 for 9 h resulted in the highest levels of aneuploidy; however, no cells were observed in the subG1 phase, which indicated the absence of apoptotic cell death. Exposure to an ELF-MF alone (1 mT or 2 mT) did not affect the percentages of G2/M-arrested cells, aneuploid cells, or the populations of cells in the subG1 phase. Moreover, when cells were exposed to a 1 mT or 2 mT ELF-MF in combination with IR (0.5 Gy) or H2O2 (0.05 mM), the ELF-MF did not further increase the percentages of G2/M-arrested cells or aneuploid cells. These results suggest that ELF-MFs alone do not induce either G2/M arrest or aneuploidy, even when administered in combination with different stressors.

A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

PubMed

Liao, Ningbo; Sun, Liang; Chen, Jiang; Zhong, Jianjun; Zhang, Yanjun; Zhang, Ronghua

2017-03-01

Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata , hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata . It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC 50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

PubMed

Chao, Xu; Zhou, Xiaojun; Zheng, Gang; Dong, Changhu; Zhang, Wei; Song, Xiaomei; Jin, Tianbo

2014-05-01

Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong's Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture. This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells. Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot. Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123 µmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins. Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.

YC-1 induces G0/G1 phase arrest and mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells.

PubMed

Lee, Miau-Rong; Lin, Chingju; Lu, Chi-Cheng; Kuo, Sheng-Chu; Tsao, Je-Wei; Juan, Yu-Ning; Chiu, Hong-Yi; Lee, Fang-Yu; Yang, Jai-Sing; Tsai, Fuu-Jen

2017-06-01

Oral cancer is a serious and fatal disease. Cisplatin is the first line of chemotherapeutic agent for oral cancer therapy. However, the development of drug resistance and severe side effects cause tremendous problems clinically. In this study, we investigated the pharmacologic mechanisms of YC-1 on cisplatin-resistant human oral cancer cell line, CAR. Our results indicated that YC-1 induced a concentration-dependent and time-dependent decrease in viability of CAR cells analyzed by MTT assay. Real-time image analysis of CAR cells by IncuCyte™ Kinetic Live Cell Imaging System demonstrated that YC-1 inhibited cell proliferation and reduced cell confluence in a time-dependent manner. Results from flow cytometric analysis revealed that YC-1 promoted G 0 /G 1 phase arrest and provoked apoptosis in CAR cells. The effects of cell cycle arrest by YC-1 were further supported by up-regulation of p21 and down-regulation of cyclin A, D, E and CDK2 protein levels. TUNEL staining showed that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G 0 /G 1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future. © Author(s) 2017. This article is published with open access by China Medical University.

Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F.

PubMed

Loftus, Kyle M; Cui, Heying; Coutavas, Elias; King, David S; Ceravolo, Amanda; Pereiras, Dylan; Solmaz, Sozanne R

2017-08-03

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

Visualizing Vpr-Induced G2 Arrest and Apoptosis

PubMed Central

Murakami, Tomoyuki; Aida, Yoko

2014-01-01

Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1) with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2). The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to characterize the

Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell.

PubMed

Kavitha, Nowroji; Ein Oon, Chern; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

2017-04-06

Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. MDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G 0 /G 1 and G 2 /M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆ Ψm ) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G 0 /G 1 and G 2 /M-phases cell cycle arrest by p53-mediated mechanism. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Anti-proliferative effects, cell cycle G2/M phase arrest and blocking of chromosome segregation by probimane and MST-16 in human tumor cell lines

PubMed Central

Lu, Da Yong; Huang, Min; Xu, Cheng Hui; Yang, Wei Yi; Hu, Chao Xin; Lin, Li Ping; Tong, Lin Jiang; Li, Mei Hong; Lu, Wei; Zhang, Xiong Wen; Ding, Jian

2005-01-01

Background Anticancer bisdioxopiperazines, including ICRF-154, razoxane (Raz, ICRF-159) and ICRF-193, are a family of anticancer agents developed in the UK, especially targeting metastases of neoplasms. Two other bisdioxopiperazine derivatives, probimane (Pro) and MST-16, were synthesized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. Cytotoxic activities and mechanisms of Raz (+)-steroisomer (ICRF-187, dexrazoxane), Pro and MST-16 against tumor cells were evaluated by MTT colorimetry, flow cytometry and karyotyping. Results Pro was cytotoxic to human tumor cell lines in vitro (IC50<50 μM for 48 h). Four human tumor cell lines (SCG-7901, K562, A549 and HL60) were susceptible to Pro at low inhibitory concentrations (IC50 values < 10 μM for 48 h). Although the IC50 against HeLa cell line of vincristine (VCR, 4.56 μM), doxorubicin (Dox, 1.12 μM) and 5-fluoruouracil (5-Fu, 0.232 μM) are lower than Pro (5.12 μM), ICRF-187 (129 μM) and MST-16 (26.4 μM), VCR, Dox and 5-Fu shows a low dose-related – high cytotoxic activity. Time-response studies showed that the cytotoxic effects of Pro are increased for 3 days in human tumor cells, whereas VCR, Dox and 5-Fu showed decreased cytotoxic action after 24 h. Cell cycle G2/M phase arrest and chromosome segregation blocking by Pro and MST-16 were noted. Although there was similar effects of Pro and MST-16 on chromosome segregation blocking action and cell cycle G2/M phase arrest at 1- 4 μM, cytotoxicity of Pro against tumor cells was higher than that of MST-16 in vitro by a factor of 3- 10 folds. Our data show that Pro may be more effective against lung cancer and leukemia while ICRF-187 and MST-16 shows similar IC50 values only against leukemia. Conclusion It suggests that Pro has a wider spectrum of cytotoxic effects against human tumor cells than other bisdioxopiperazines, especially against solid tumors, and with a single cytotoxic pathway of Pro and MST-16 affecting

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids.

PubMed

da Silva, Marcelo Santos; Muñoz, Paula Andrea Marin; Armelin, Hugo Aguirre; Elias, Maria Carolina

2017-11-01

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis.

PubMed

Li, Huiyan; Peng, Xuan; Wang, Yating; Cao, Shirong; Xiong, Liping; Fan, Jinjin; Wang, Yihan; Zhuang, Shougang; Yu, Xueqing; Mao, Haiping

2016-09-01

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G2/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G2/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G2/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G1/S phase, and increased COL1 expression in the G2/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G2/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G2/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G2/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G2/M arrest.

Expression of programmed cell death1 in T follicular helper cells is regulated by prostaglandin E2 secreted by HBV-infected HepG2.2.1.5 cells.

PubMed

Sui, Zhefeng; Shi, Ying; Gao, Zhiling; Yang, Deguang; Wang, Zhihao

2017-06-01

The present study aimed to investigate the distribution of T follicular helper (Tfh)-cell subsets in patients with hepatitis B virus (HBV) and determine the underlying mechanism of HBV regulation of Tfh cells. The frequency of peripheral blood Tfh subsets was analyzed using flow cytometry. The expression level of programmed cell death‑1 (PD‑1) and prostaglandin E2 (PGE2) was quantified using reverse transcription‑quantitative polymerase chain reaction and western blotting. The PGE2 level in culture supernatant was detected using enzyme‑linked immunosorbent assay. A Transwell chamber was used to co‑culture Tfh cells with HepG2 and HepG2.2.1.5. The percentage of inducible T‑cell costimulator (ICOS)+ and total Tfh cells was high at the immune activation (IA) group; however, it was reduced in the immune tolerance (IT), responders with HBsAg seroconversion (RP) and healthy control (HC) groups. The percentage of PD‑1+ Tfh cells was significantly higher in IA and IT compared with RP and HC. The ratio of PD‑1+/total Tfh cells was positively correlated with the load of HBV DNA; therefore, this ratio may act as an indicator for HBV replication. The expression level of PD‑1 in Tfh cells was higher in the HepG2.2.1.5 co‑cultured group compared with the HepG2 group, this may be due to the high PGE2 expression level in HBV‑infected HepG2.2.1.5 cells. The findings of the present study revealed an imbalanced distribution of PD‑1+ Tfh cells in patients with HBV at different immune phases. Additionally, HBV may upregulate the expression of PD‑1 in Tfh cells by promoting HepG2.2.1.5 to secret PGE2. Identifying the effect of HBV on Tfh‑cell subsets is crucial for improving immuno-based therapy for HBV.

Eupatilin, a dietary flavonoid, induces G2/M cell cycle arrest in human endometrial cancer cells.

PubMed

Cho, Jung-Hoon; Lee, Jong-Gyu; Yang, Yeong-In; Kim, Ji-Hyun; Ahn, Ji-Hye; Baek, Nam-In; Lee, Kyung-Tae; Choi, Jung-Hye

2011-08-01

This study is the first to investigate the antiproliferative effect of eupatilin in human endometrial cancer cells. Eupatilin, a naturally occurring flavonoid isolated from Artemisia princeps, has anti-inflammatory, anti-oxidative, and anti-tumor activities. In the present study, we investigated the potential effect of eupatilin on cell growth and its molecular mechanism of action in human endometrial cancer cells. Eupatilin was more potent than cisplatin in inhibiting cell viability in the human endometrial cancer cell lines Hec1A and KLE. Eupatilin showed relatively low cytotoxicity in normal human endometrial cells HES and HESC cells when compared to cisplatin. Eupatilin induced G2/M phase cell cycle arrest in a time- and dose-dependent manner, as indicated by flow cytometry analysis. In addition, treatment of Hec1A cells with eupatilin resulted in a significant increase in the expression of p21(WAF1/CIP1) and in the phosphorylation of Cdc25C and Cdc2. Knockdown of p21 using specific siRNAs significantly compromised eupatilin-induced cell growth inhibition. Interestingly, levels of mutant p53 in Hec1A cells decreased markedly upon treatment with eupatilin, and p53 siRNA significantly increased p21 expression. Moreover, eupatilin modulated the phosphorylation of protein kinases ERK1/2, Akt, ATM, and Chk2. These results suggest that eupatilin inhibits the growth of human endometrial cancer cells via G2/M phase cell cycle arrest through the up-regulation of p21 by the inhibition of mutant p53 and the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway. Copyright © 2011 Elsevier Ltd. All rights reserved.

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

PubMed Central

2013-01-01

Background Depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear. Methods Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo. Results We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model. Conclusion Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma. PMID:23578185

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest.

PubMed

Li, Guilin; Zhang, Zhenxing; Wang, Renzhi; Ma, Wenbin; Yang, Ying; Wei, Junji; Wei, Yanping

2013-04-11

Depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear. Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo. We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model. Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2

PubMed Central

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E.; Chen, Shuang; Bodie, Wesley W.; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A.; Kramer, Lora B.; Dent, Paul

2011-01-01

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition. PMID:21911831

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2.

PubMed

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E; Chen, Shuang; Bodie, Wesley W; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A; Kramer, Lora B; Dent, Paul; Grant, Steven

2011-11-10

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype.

PubMed

Miglionico, Rocchina; Ostuni, Angela; Armentano, Maria Francesca; Milella, Luigi; Crescenzi, Elvira; Carmosino, Monica; Bisaccia, Faustino

2017-01-01

Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype. Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype. To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21 Cip p53 independent; and 4) downregulation of lamin A/C. These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.

The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase.

PubMed

Cortes-Bratti, X; Chaves-Olarte, E; Lagergård, T; Thelestam, M

1999-01-01

The potent cytolethal distending toxin produced by Haemophilus ducreyi is a putative virulence factor in the pathogenesis of chancroid. We studied its action on eukaryotic cells, with the long-term goal of understanding the pathophysiology of the disease. Intoxication of cultured human epithelial-like cells, human keratinocytes, and hamster fibroblasts was irreversible, and appeared as a gradual distention of three- to fivefold the size of control cells. Organized actin assemblies appeared concomitantly with cell enlargement, promoted by a mechanism that probably does not involve small GTPases of the Rho protein family. Intoxicated cells did not proliferate. Similar to cells treated with other cytolethal distending toxins, these cells accumulated in the G2 phase of the cell cycle, demonstrating an increased level of the tyrosine phosphorylated (inactive) form of the cyclin-dependent kinase p34(cdc2). DNA synthesis was not affected until several hours after this increase, suggesting that the toxin acts directly on some kinase/phosphatase in the signaling network controlling the p34(cdc2) activity. We propose that this toxin has an important role both in the generation of chancroid ulcers and in their slow healing. The toxin may also be an interesting new tool for molecular studies of the eukaryotic cell- cycle machinery.

The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase

PubMed Central

Cortes-Bratti, Ximena; Chaves-Olarte, Esteban; Lagergård, Teresa; Thelestam, Monica

1999-01-01

The potent cytolethal distending toxin produced by Haemophilus ducreyi is a putative virulence factor in the pathogenesis of chancroid. We studied its action on eukaryotic cells, with the long-term goal of understanding the pathophysiology of the disease. Intoxication of cultured human epithelial-like cells, human keratinocytes, and hamster fibroblasts was irreversible, and appeared as a gradual distention of three- to fivefold the size of control cells. Organized actin assemblies appeared concomitantly with cell enlargement, promoted by a mechanism that probably does not involve small GTPases of the Rho protein family. Intoxicated cells did not proliferate. Similar to cells treated with other cytolethal distending toxins, these cells accumulated in the G2 phase of the cell cycle, demonstrating an increased level of the tyrosine phosphorylated (inactive) form of the cyclin-dependent kinase p34cdc2. DNA synthesis was not affected until several hours after this increase, suggesting that the toxin acts directly on some kinase/phosphatase in the signaling network controlling the p34cdc2 activity. We propose that this toxin has an important role both in the generation of chancroid ulcers and in their slow healing. The toxin may also be an interesting new tool for molecular studies of the eukaryotic cell- cycle machinery. PMID:9884340

Hyperglycemia and anthocyanin inhibit quercetin metabolism in HepG2 cells

USDA-ARS?s Scientific Manuscript database

A high glucose (Glu) milieu promotes generation of reactive oxygen species, which may not only cause cellular damage, but also modulate phase II enzymes that are responsible for the metabolism of flavonoids. Thus, we examined the effect of a high Glu milieu on quercetin (Q) metabolism in HepG2 cells...

Ursolic acid sensitizes cisplatin-resistant HepG2/DDP cells to cisplatin via inhibiting Nrf2/ARE pathway

PubMed Central

Wu, Shouhai; Zhang, Tianpeng; Du, Jingsheng

2016-01-01

Background Combinations of adjuvant sensitizers with anticancer drugs is a promising new strategy to reverse chemoresistance. Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds known to have many pharmacological characteristics such as anti-inflammatory and anticancer properties. This study investigates whether UA can sensitize hepatocellular carcinoma cells to cisplatin. Materials and methods Cells were transfected with nuclear factor erythroid-2-related factor 2 (Nrf2) small interfering RNA and Nrf2 complementary DNA by using Lipofectin 2000. The cytotoxicity of cells was investigated by Cell Counting Kit 8 assay. Cell apoptosis, cell cycle, reactive oxygen species, and mitochondrial membrane potential were detected by flow cytometry fluorescence-activated cell sorting. The protein level of Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), and heme oxygenase-1 (HO-1) was detected by Western blot analysis. Results The results showed that the reverse index was 2.9- and 9.69-fold by UA of 1.125 μg/mL and 2.25 μg/mL, respectively, for cisplatin to HepG2/DDP cells. UA–cisplatin combination induced cell apoptosis and reactive oxygen species, blocked the cell cycle in G0/G1 phase, and reduced the mitochondrial membrane potential. Mechanistically, UA–cisplatin dramatically decreased the expression of Nrf2 and its downstream genes. The sensibilization of UA–cisplatin combination was diminished in Nrf2 small interfering RNA-transfected HepG2/DDP cells, as well as in Nrf2 complementary DNA-transfected HepG2/DDP cells. Conclusion The results confirmed the sensibilization of UA on HepG2/DDP cells to cisplatin, which was possibly mediated via the Nrf2/antioxidant response element pathway. PMID:27822011

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen.

PubMed

Wang, Jianling; Wang, Gangduo; Khan, M Firoze

2015-01-01

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a

Apoptosis Induction by Polygonum minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and S-phase cell cycle arrest in HepG2 cell line.

PubMed

Mohd Ghazali, Mohd Alfazari; Al-Naqeb, Ghanya; Krishnan Selvarajan, Kesavanarayanan; Hazizul Hasan, Mizaton; Adam, Aishah

2014-01-01

Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1-F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects.

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc- cells

NASA Technical Reports Server (NTRS)

Hughes-Fulford, M.

1994-01-01

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the -49 lymphoma variant (cyc-) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A1 (dmPGA1) inhibits DNA synthesis and cell growth in cyc- cells. DNA synthesis is inhibited 42% by dmPGA1 (50 microM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the alpha, beta unsaturated ketone ring. Dimethyl PGA1 is most effective in inhibiting DNA synthesis in cyc- cells, with prostaglandins PGE1 and PGB1 being less potent inhibitors of DNA synthesis. DmPGE2 caused a significant stimulation of DNA synthesis. S-49 cyc- variant cells exposed to (30-50 microns) dmPGA1, arrested in the G1 phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc- cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G1, S, G2, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G1 cell cycle block.(ABSTRACT TRUNCATED AT 250 WORDS).

Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells.

PubMed

Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-02-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60-75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G 0 /G 1 phase and reduced the number of cells in the S phase, as compared with the control group (P<0.05). Western blot analysis demonstrated that arctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G 0 /G 1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas.

Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells

PubMed Central

Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-01-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60–75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G0/G1 phase and reduced the number of cells in the S phase, as compared with the control group (P<0.05). Western blot analysis demonstrated that arctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G0/G1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas. PMID:28356992

Susceptibility of Hep3B cells in different phases of cell cycle to tBid.

PubMed

Ma, Shi-Hong; Chen, George G; Ye, Caiguo; Leung, Billy C S; Ho, Rocky L K; Lai, Paul B S

2011-01-01

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC. Copyright © 2010 Elsevier B.V. All rights reserved.

G protein-coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis.

PubMed

Mori, Taisuke; Ito, Fumitake; Matsushima, Hiroshi; Takaoka, Osamu; Tanaka, Yukiko; Koshiba, Akemi; Kusuki, Izumi; Kitawaki, Jo

2015-05-01

To demonstrate the effects of the selective G protein-coupled estrogen receptor 1 (GPER) agonist G-1 in human ovarian endometriotic stromal cells (ESCs). Experimental in vitro study. University hospital. A total of 33 patients with ovarian endometrioma. Endometriotic stromal cells from ovarian chocolate cysts were treated with the GPER agonist G-1. The primary outcomes were cell proliferation, measured using the WST-8 assay; cell cycle, as analyzed using flow cytometry, fluorescent immunocytochemistry, and cytotoxicity; caspase activity, as measured by fluorescent and luminescent enzyme assays; and protein expression levels, as determined by Western blot analysis. G-1 suppressed ESC proliferation in a concentration-dependent manner. The inhibitory effect was not blocked when GPER signaling pathways, including the GPER itself, were inhibited. G-1 induced cell cycle arrest and accumulation in the sub-G1 phase in ESCs. Immunofluorescence analysis demonstrated that G-1 interrupted microtubule assembly at the mitotic phase. G-1 also induced caspase-3-dependent apoptosis without significant cytotoxicity. G-1 suppressed proliferation and induced apoptosis in ESCs, suggesting the potential use of this compound as a therapeutic drug for the treatment of endometriosis. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Dehydrozingerone, a structural analogue of curcumin, induces cell-cycle arrest at the G2/M phase and accumulates intracellular ROS in HT-29 human colon cancer cells.

PubMed

Yogosawa, Shingo; Yamada, Yasumasa; Yasuda, Shusuke; Sun, Qi; Takizawa, Kaori; Sakai, Toshiyuki

2012-12-28

Dehydrozingerone (1) is a pungent constituent present in the rhizomes of ginger (Zingiber officinale) and belongs structurally to the vanillyl ketone class. It is a representative of half the chemical structure of curcumin (2), which is an antioxidative yellow pigment obtained from the rhizomes of turmeric (Curcuma longa). Numerous studies have suggested that 2 is a promising phytochemical for the inhibition of malignant tumors, including colon cancer. On the other hand, there have been few studies on the potential antineoplastic properties of 1, and its mode of action based on a molecular mechanism is little known. Therefore, the antiproliferative effects of 1 were evaluated against HT-29 human colon cancer cells, and it was found that 1 dose-dependently inhibited growth at the G2/M phase with up-regulation of p21. Dehydrozingerone additionally led to the accumulation of intracellular ROS, although most radical scavengers could not clearly repress the cell-cycle arrest at the G2/M phase. Furthermore, two synthetic isomers of 1 (iso-dehydrozingerone, 3, and ortho-dehydrozingerone, 4) were also examined. On comparing of their activities, accumulation of intracellular ROS was found to be interrelated with growth-inhibitory effects. These results suggest that analogues of 1 may be potential chemotherapeutic agents for colon cancer.

The ethanol extract from Artemisia princeps Pampanini induces p53-mediated G1 phase arrest in A172 human neuroblastoma cells.

PubMed

Park, Eun Young; Lee, Kyung-Won; Lee, Heon-Woo; Cho, Young-Wuk; Baek, Nam-In; Chung, Hae-Gon; Jeong, Tae-Sook; Choi, Myung-Sook; Lee, Kyung-Tae

2008-06-01

In the present study, the antiproliferative effects of the ethanol extract of Artemisia princeps Pampanini (EAPP) and the mechanism involved were investigated. Of the various cancer cells examined, human neuroblastoma A172 cells were most sensitive to EAPP, and their proliferation was dose- and time-dependently inhibited by EAPP. DNA flow cytometry analysis indicated that EAPP notably induced the G(1) phase arrest in A172 cells. Of the G(1) phase cycle-related proteins examined, the expressions of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 and of cyclin D(1), D(2), and D(3) were found to be markedly reduced by EAPP, whereas cyclin E was unaffected. Moreover, the protein and mRNA levels of the CDK inhibitors p16(INK4a), p21(CIP1/WAF1), and p27(KIP1) were increased, and the activities of CDK2, CDK4, and CDK6 were reduced. Furthermore, the expressions of E2F-1 and of phosphorylated pRb were also decreased, and the protein levels of p53 and pp53 (Ser15) were increased. Up-regulation of p21(CIP1/WAF1) was found to be mediated by a p53-dependent pathway in EAPP-induced G(1)-arrested A172 cells. When these data are taken together, the EAPP was found to potently inhibit the proliferation of human neuroblastoma A172 cells via G(1) phase cell cycle arrest.

Apoptosis Induction by Polygonum minus Is Related to Antioxidant Capacity, Alterations in Expression of Apoptotic-Related Genes, and S-Phase Cell Cycle Arrest in HepG2 Cell Line

PubMed Central

Mohd Ghazali, Mohd Alfazari; Al-Naqeb, Ghanya; Krishnan Selvarajan, Kesavanarayanan; Hazizul Hasan, Mizaton; Adam, Aishah

2014-01-01

Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1–F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects. PMID:24955361

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

PubMed

Zhang, Yali; Guo, Zonglou; Xu, Lihong

2014-03-01

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen

PubMed Central

Wang, Jianling; Wang, Gangduo; Khan, M. Firoze

2015-01-01

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a

Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

PubMed

Kuriyama, Shigeki; Hitomi, Misuzu; Yoshiji, Hitoshi; Nonomura, Takako; Tsujimoto, Tatsuhiro; Mitoro, Akira; Akahane, Takami; Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Masaki, Tsutomu; Uchida, Naohito

2005-08-01

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

Small Molecule TH-39 Potentially Targets Hec1/Nek2 Interaction and Exhibits Antitumor Efficacy in K562 Cells via G0/G1 Cell Cycle Arrest and Apoptosis Induction.

PubMed

Zhu, Yongxia; Wei, Wei; Ye, Tinghong; Liu, Zhihao; Liu, Li; Luo, Yong; Zhang, Lidan; Gao, Chao; Wang, Ningyu; Yu, Luoting

2016-01-01

Cancer is still a major public health issue worldwide, and new therapeutics with anti-tumor activity are still urgently needed. The anti-tumor activity of TH-39, which shows potent anti-proliferative activity against K562 cells with an IC50 of 0.78 µM, was investigated using immunoblot, co-immunoprecipitation, the MTT assay, and flow cytometry. Mechanistically, TH-39 may disrupt the interaction between Hec1 and Nek2 in K562 cells. Moreover, TH-39 inhibited cell proliferation in a concentration- and time-dependent manner by influencing the morphology of K562 cells and inducing G0/G1 phase arrest. G0/G1 phase arrest was associated with down-regulation of CDK2-cyclin E complex and CDK4/6-cyclin D complex activities. Furthermore, TH-39 also induced cell apoptosis, which was associated with activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax. TH-39 could also decrease mitochondrial membrane potential (Δψm) and increase reactive oxygen species (ROS) accumulation in K562 cells. The results indicated that TH-39 might induce apoptosis via the ROS-mitochondrial apoptotic pathway. This study highlights the potential therapeutic efficacy of the anti-cancer compound TH-39 in treatment-resistant chronic myeloid leukemia. © 2016 The Author(s) Published by S. Karger AG, Basel.

Chromosomal radiosensitivity in G{sub 2}-phase lymphocytes as an indicator of cancer predisposition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, D.; Spreadborough, A.R.; Jones, L.A.

Sanford et al. have reported that G{sub 2}-phase cells from many heritable cancer-prone conditions exhibit higher yields of X-ray-induced chromosome damage than those found in the majority of healthy controls. We have applied their protocol to lymphocytes of a group of control and cancer-prone individuals to see if we could confirm these observations. For control donors we observed higher aberration yields, different kinetics and more interexperiment variability than found by Sanford et al. These differences could not be attributed to unavoidable minor variations in procedures (e.g. serum batches, glassware washing methods), but the difference in X-ray qualities used in themore » two laboratories may have made a small contribution to the discrepancies. We attribute some of our experimental variability to the fact that, to varying extents in different experiments, centrifugation of cells prior to irradiation can slow down the progression of cells into metaphase and that cells can continue to repair during the harvesting procedure (centrifugation and hypotonic treatment). We have applied the assay to cases of ataxia telangiectasia (AT, homozygotes and heterozygotes), xeroderma pigmentosum (homozygotes and heterozygotes), familiar adenomatous polyposis and the syndromes Li-Fraumeni, basal cell nevus, Down`s and Fanconi`s but have been unable to discriminate between these groups and controls except for AT homozygotes. By including a control sample in parallel with samples from cancer-prone groups we found a significant difference in mean aberration yields between controls and AT homozygotes and heterozygotes, but not for the other groups. Since technical features could explain the discrepancies between our laboratories, we have devised our own G{sub 2}-phase assay which appears to be giving promising results. 35 refs., 24 figs., 6 tabs.« less

Tributyltin induces cell cycle arrest at G1 phase in the yeast Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Akiyama, Koichi; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

2014-04-01

Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

53BP1 promotes microhomology-mediated end-joining in G1-phase cells

PubMed Central

Xiong, Xiahui; Du, Zhanwen; Wang, Ying; Feng, Zhihui; Fan, Pan; Yan, Chunhong; Willers, Henning; Zhang, Junran

2015-01-01

Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair. PMID:25586219

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

PubMed

Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

1984-11-01

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

Action of caffeine on x-irradiated HeLa cells. V. Identity of the sector of cells that expresses potentially lethal damage in G/sub 1/ and G/sub 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beetham, K.L.; Tolmach, L.J.

1982-07-01

When HeLa S3 cells are irradiated in early G/sub 1/ with 4 Gy of 220-kV x rays and are then incubated in growth medium containing up to 5 mM caffeine, survival is reduced (as reported previously), reaching a concentration-dependent plateau. Cell killing presumably occurs as a result of the fixation of a portion of the potentially lethal damage the cells contain. These cells respond to continued treatment with caffeine at concentrations greater than 2 mM during S, but less so than during G/sub 1/. When they reach G/sub 2/ arrest, however, extensive cell killing again occurs (reported previously), presumably alsomore » the result of potentially lethal damage fixation. G/sub 1/-irradiated cultures that are treated with caffeine either continuously at a concentration in the range 1 to 5 mM, or at 10 mM for 8 hr and subsequently with the low concentration, achieve the same survival level in G/sub 2/, provided that the potentially lethal damage is not repaired during G/sub 1/ and S. Repair seems to be completely inhibited in the presence of 3 to 4 mM caffeine. The results indicate that fixation of potentially lethal damage occurs in the same sector of cells in G/sub 1/ and G/sub 2/, suggesting that the same cellular lesion gives rise to cell killing in the two phases.« less

The Bone-specific Expression of Runx2 Oscillates during the Cell Cycle to Support a G1-related Antiproliferative Function in Osteoblasts*

PubMed Central

Galindo, Mario; Pratap, Jitesh; Young, Daniel W.; Hovhannisyan, Hayk; Im, Hee-Jeong; Choi, Je-Yong; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; van Wijnen, Andre J.

2010-01-01

The Runx2 (CBFA1/AML3/PEBP2αA) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of Runx2 gene transcription, mRNA and protein, are each up-regulated with cessation of cell growth (i.e. G0/G1 transition) in preconfluent MC3T3 osteoblastic cells that do not yet express mature bone phenotypic gene expression. Cell growth regulation of Runx2 is also observed in primary calvarial osteoblasts and other osteoblastic cells with relatively normal cell growth characteristics, but not in osteosarcoma cells (e.g. SAOS-2 and ROS17/2.8). Runx2 levels are cell cycle-regulated in MC3T3 cells with respect to the G1/S and M/G1 transitions: expression oscillates from maximal levels during early G1 to minimal levels during early S phase and mitosis. However, in normal or immortalized (e.g. ATDC5) chondrocytic cells, Runx2 expression is suppressed during quiescence, and Runx2 levels are not regulated during G1 and S phase in ATDC5 cells. Antisense or small interfering RNA-mediated reduction of the low physiological levels of Runx2 in proliferating MC3T3 cells does not accelerate cell cycle progression. However, forced expression of Runx2 suppresses proliferation of MC3T3 preosteoblasts or C2C12 mesenchymal cells which have osteogenic potential. Forced elevation of Runx2 in synchronized MC3T3 cells causes a delay in G1. We propose that Runx2 levels and function are biologically linked to a cell growth-related G1 transition in osteoblastic cells. PMID:15781466

Anethum graveolens (dill) - A medicinal herb induces apoptosis and cell cycle arrest in HepG2 cell line.

PubMed

Mohammed, Furkhan Ahmed; Elkady, Ayman I; Syed, Fareeduddin Quadri; Mirza, Muqtadir Baig; Hakeem, Khalid Rehman; Alkarim, Saleh

2018-06-12

The medicinal herb, Anethum graveolens L. (dill) is one of the potent culinary herbs used as an alternative form of medicine worldwide. The unguent topical Oil from the aerial parts of A. graveolens was found to be effective in the management of uterus cancer in ethnomedicine has been reported. The incidence and mortality rates of Hepatocellular carcinoma (HCC) are steadily rising worldwide, especially, in underdeveloped and developing countries. Moreover, HCC develops rapidly in patients with chronic cirrhosis or hepatitis, where the solid tumours/malignancies coexist with the inflammation. Recent studies have shown that the medicinal herb, Anethum graveolens, holds anticancer potential, which could be a promising approach for the treatment of various tumours. In the current study, we have analysed the antiproliferative effect of ethyl acetate fraction of Dill Seeds (EAFD) on HepG2 cell line. Cell viability and proliferation were observed by MTT assay; Morphological changes were studied using fluorescent stains like Hoechst 33342, acridine orange/ethidium bromide and JC-1 dye. Further, the pro-apoptotic activity was demonstrated through Annexin-V-FITC/ PI assay and cell cycle analysis. Different concentrations (0.1, 0.2, 0.4, 0.6, 0.8 mg/ml) of EAFD were studied. EAFD markedly suppressed the proliferation of HepG2 cells in a dose and time-dependent manner. The phase contrast and fluorescence microscopy revealed the morphological alterations like disruption, shrinkage, detachment and blebbing of cell membrane accompanied by nuclear condensation after exposure to EAFD. Radical scavenging activity was evidenced by measurement of ROS levels post-treatment. Modulation of mitochondrial membrane potential was exhibited leading to the activation of caspases 3/7 and 9 which is a committed step towards apoptosis. Annexin V-FITC/ PI assay and cell cycle, later confirmed the apoptosis and cell cycle arrest in 'G2/M' phase through flow cytometric analysis. In conclusion, a

A Cyclin D2-derived peptide acts on specific cell cycle phases by activating ERK1/2 to cause the death of breast cancer cells.

PubMed

Russo, Lilian C; Araujo, Christiane B; Iwai, Leo K; Ferro, Emer S; Forti, Fabio L

2017-01-16

Protein degradation by the proteasome generates functional intracellular peptides. Pep5, a peptide derived from Cyclin D2, induces cell death in tumor cell lines and reduces the volume of rat C6 glioblastoma tumors in vivo. Here, we chose the human MDA-MB-231 breast cancer cells to evaluate the mechanism of cell death induced by pep5 in different phases of the cell cycle. Fluorescently labeled pep5, monitored by real time confocal microscopy, entered the MDA-MB-231 cells 3min after application and localized to the nucleus and cytoplasm. Pep5-induced cell death was increased when the MDA-MB-231 cell population was arrested at the G1/S transition or in S phase compared to asynchronous cells. Pep5 induced permanent extracellular signal-regulated kinase (ERK1/2) phosphorylation in MDA-MB-231 cells synchronized in G1/S or S phase. Affinity chromatography followed by mass spectrometry identified CLIC1 and Plectin as the only two proteins that interacted with pep5 in both asynchronous and synchronized MDA-MB-231 cells. These interactions could explain the long-lasting ERK1/2 phosphorylation and the cytoskeleton perturbations in the MDA-MB-231 cells, in which the stress fibers' integrity is affected by pep5 treatments. These data suggest that pep5 has potential therapeutic properties for treating specific types of cancers, such as breast cancer cells. Pep5, a natural intracellular peptide formed by the degradation of Cyclin D2 through the ubiquitin-proteasome system, induces cell death when reintroduced into MDA-MB-231 breast cancer cells, which express low levels of Cyclin D2, specifically in G1/S arrested cells or in cells that are passing through S phase. Under these conditions, pep5 is able to interact with different intracellular proteins, primarily cytoskeleton and proteasome components, which can lead to cellular apoptosis. Together, our data suggest that pep5 is an intracellular peptide with therapeutic potential for treating specific types of tumors with low

NF-κB mediates the antiproliferative and proapoptotic effects of bergamot juice in HepG2 cells.

PubMed

Ferlazzo, Nadia; Cirmi, Santa; Russo, Marina; Trapasso, Elena; Ursino, Maria Rita; Lombardo, Giovanni Enrico; Gangemi, Sebastiano; Calapai, Gioacchino; Navarra, Michele

2016-02-01

Among cancers, hepatocellular carcinoma is one of the commonest worldwide, and its incidence is increasing around the world. A lot of evidence underlines that natural substances usually consumed in the diet can have an important role in the prevention of cancer. In this study we investigated the molecular mechanisms underlying the antiproliferative activity of Citrus bergamia (bergamot) juice (BJ) in human hepatocellular carcinoma HepG2 cells. HepG2 cells were exposed to BJ and then cell proliferation, cell cycle progression, apoptosis and NF-κB nuclear translocation were evaluated. Here we present results demonstrating that BJ reduced the growth rate of human hepatocellular carcinoma HepG2 cells in a time- and concentration-dependent manner, by a mechanism involving the activation of apoptotic machinery via both intrinsic and extrinsic pathways. Moreover, BJ increased expression of P53 and P21 proteins that may be responsible for the HepG2 cell cycle arrest in G2 phase. In addition, BJ reduced NF-κB nuclear translocation. Our data demonstrate the ability of BJ in reducing the growth of HepG2 cells, revealing its mechanism of action and suggesting a promising role as anticancer drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

PubMed

Ding, Xia; He, Zhuohao; Zhou, Kechun; Cheng, Ju; Yao, Hailan; Lu, Dongliang; Cai, Rong; Jin, Yening; Dong, Bin; Xu, Yinghui; Wang, Yizheng

2010-07-21

Patients with glioblastoma multiforme, the most aggressive form of glioma, have a median survival of approximately 12 months. Calcium (Ca(2+)) signaling plays an important role in cell proliferation, and some members of the Ca(2+)-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of many types of cancer cells. In this study, we investigated the role of TRPC6 in cell cycle progression and in the development of human glioma. TRPC6 protein and mRNA expression were assessed in glioma (n = 33) and normal (n = 17) brain tissues from patients and in human glioma cell lines U251, U87, and T98G. Activation of TRPC6 channels was tested by platelet-derived growth factor-induced Ca(2+) imaging. The effect of inhibiting TRPC6 activity or expression using the dominant-negative mutant TRPC6 (DNC6) or RNA interference, respectively, was tested on cell growth, cell cycle progression, radiosensitization of glioma cells, and development of xenografted human gliomas in a mouse model. The green fluorescent protein (GFP) and wild-type TRPC6 (WTC6) were used as controls. Survival of mice bearing xenografted tumors in the GFP, DNC6, and WTC6 groups (n = 13, 15, and 13, respectively) was compared using Kaplan-Meier analysis. All statistical tests were two-sided. Functional TRPC6 was overexpressed in human glioma cells. Inhibition of TRPC6 activity or expression attenuated the increase in intracellular Ca(2+) by platelet-derived growth factor, suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 activation and cell division cycle 25 homolog C expression regulated the cell cycle arrest. Inhibition of TRPC6 activity also reduced tumor volume in a subcutaneous mouse model of xenografted human tumors (P = .014 vs GFP; P < .001 vs WTC6) and increased mean survival in mice in an intracranial

The Cak1p Protein Kinase Is Required at G(1)/S and G(2)/M in the Budding Yeast Cell Cycle

PubMed Central

Sutton, A.; Freiman, R.

1997-01-01

The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G(2) to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G(2) to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G(1) into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function. PMID:9286668

Investigating free radical generation in HepG2 cells using immuno-spin trapping.

PubMed

Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Kawazoe, Kazuyoshi; Tsuchiya, Koichiro; Tamaki, Toshiaki; Mason, Ronald P

2014-10-01

Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity. Copyright © 2014. Published by Elsevier Inc.

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

PubMed Central

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

The color and size of chili peppers (Capsicum annuum) influence Hep-G2 cell growth.

PubMed

Popovich, David G; Sia, Sharon Y; Zhang, Wei; Lim, Mon L

2014-11-01

Four types of chili (Capsicum annuum) extracts, categorized according to color; green and red, and size; small and large were studied in Hep-G2 cells. Red small (RS) chili had an LC50 value of 0.378 ± 0.029 compared to green big (GB) 1.034 ± 0.061 and green small (GS) 1.070 ± 0.21 mg/mL. Red big (RB) was not cytotoxic. Capsaicin content was highest in RS and produced a greater percentage sub-G1 cells (6.47 ± 1.8%) after 24 h compared to GS (2.96 ± 1.3%) and control (1.29 ± 0.8%) cells. G2/M phase was reduced by GS compared to RS and control cells. RS at the LC50 concentration contained 1.6 times the amount of pure capsaicin LC50 to achieve the same effect of capsaicin alone. GS and GB capsaicin content at the LC50 value was lower (0.2 and 0.66, respectively) compared to the amount of capsaicin to achieve a similar reduction in cell growth.

Effect of baicalin-copper on the induction of apoptosis in human hepatoblastoma cancer HepG2 cells.

PubMed

Li, Xiaoli; Zou, Kaili; Gou, Jing; Du, Qin; Li, Dejuan; He, Xiaoyan; Li, Zhubo

2015-03-01

The medical properties of baicalin have been well known for many years. However, the discovery that baicalin in the presence of metal ions is more effective than baicalin alone changed the course of drug research. The present study was designed to investigate the effect and possible mechanism of apoptosis induced by baicalin-copper in a human hepatoblastoma cancer cell line (HepG2) and in vivo. This study demonstrated that baicalin-copper suppresses the proliferation of HepG2 cells in a dose-dependent manner. Intraperitoneal injection of baicalin-copper resulted in a significant decrease in tumor growth in xenografts in nude mice. Acridine orange staining and flow cytometry analysis demonstrated that baicalin-copper induced apoptosis in HepG2 cells and caused cells to arrest in G2-M phase of the cell cycle. Furthermore, baicalin-copper treatment significantly increased the Bax/Bcl-2 ratio and p38 levels, as well as decreased the expression of caspase-3, p-PI3K, p-Akt and p-mTOR (P < 0.01). All of the evidences above indicate that baicalin-copper induces apoptosis in HepG2 cells by down-regulating the PI3K/Akt/mTOR signaling pathway.

Antioxidant and anticancer activity of Artemisia princeps var. orientalis extract in HepG2 and Hep3B hepatocellular carcinoma cells.

PubMed

Choi, Eun-Jeong; Kim, Gun-Hee

2013-10-01

The aim of the present study was to investigate antioxidant and the anticancerigen activity of a methanol extract from Artemisia princeps var. orientalis (APME), a well-known traditional herbal medicine in Asia, in hepatocellular cancer cells. To evaluate the antioxidant activity of APME, reactive oxygen species (ROS) and the antioxidant enzymes, superoxide dismutase (SOD) and catalase were investigated in HepG2 cells exposed to APME (5, 100, and 200 µg/mL) for 72 h. Then, to evaluate the anticancer activity of APME, we investigated the proliferation and apoptosis induction of HepG2 and Hep3B cells exposed to APME (1-200 µg/mL) for 24, 48, and 72 h. APME dose-dependently reduced the generation of ROS in the presence of H2O2 compared with control cells. Furthermore, it increased catalase and SOD activity. Moreover, APME inhibited cell proliferation in a dose- and time-dependent manner, but at concentrations lower than 100 µg/mL, the inhibition was less dose-dependent than time-dependent. HepG2 and Hep3B cells exposed to 5, 100, and 200 µg/mL APME for 72 h underwent cell cycle arrest and apoptosis. Exposure to APME resulted in a significant increase in the number of cells in G1 phase and a decrease in the G2/M phase cell population. In addition, APME induced P53 expression of HepG2 cells in a dose-dependent manner, and played a role in the downregulation of Bcl-2 and upregulation of Bax in both HepG2 and Hep3B cells. These results indicate the potential role of APME as an antioxidant and anticancerigen agent in hepatocarcinoma cell lines.

Antioxidant and anticancer activity of Artemisia princeps var. orientalis extract in HepG2 and Hep3B hepatocellular carcinoma cells

PubMed Central

Choi, Eun-Jeong

2013-01-01

Objective The aim of the present study was to investigate antioxidant and the anticancerigen activity of a methanol extract from Artemisia princeps var. orientalis (APME), a well-known traditional herbal medicine in Asia, in hepatocellular cancer cells. Methods To evaluate the antioxidant activity of APME, reactive oxygen species (ROS) and the antioxidant enzymes, superoxide dismutase (SOD) and catalase were investigated in HepG2 cells exposed to APME (5, 100, and 200 µg/mL) for 72 h. Then, to evaluate the anticancer activity of APME, we investigated the proliferation and apoptosis induction of HepG2 and Hep3B cells exposed to APME (1-200 µg/mL) for 24, 48, and 72 h. Results APME dose-dependently reduced the generation of ROS in the presence of H2O2 compared with control cells. Furthermore, it increased catalase and SOD activity. Moreover, APME inhibited cell proliferation in a dose- and time-dependent manner, but at concentrations lower than 100 µg/mL, the inhibition was less dose-dependent than time-dependent. HepG2 and Hep3B cells exposed to 5, 100, and 200 µg/mL APME for 72 h underwent cell cycle arrest and apoptosis. Exposure to APME resulted in a significant increase in the number of cells in G1 phase and a decrease in the G2/M phase cell population. In addition, APME induced P53 expression of HepG2 cells in a dose-dependent manner, and played a role in the downregulation of Bcl-2 and upregulation of Bax in both HepG2 and Hep3B cells. Conclusions These results indicate the potential role of APME as an antioxidant and anticancerigen agent in hepatocarcinoma cell lines. PMID:24255577

Cytotoxicity of mequindox and its metabolites in HepG2 cells in vitro and murine hepatocytes in vivo.

PubMed

Liu, Yingchun; Jiang, Wei; Chen, Yongjun; Liu, Yanyan; Zeng, Peng; Xue, Feiqun; Wang, Quan

2016-02-01

Mequindox, a quinoxaline 1,4-dioxide, is widely used as a feed additive in the Chinese livestock industry because of its effective antibacterial properties. Many recent studies have found that mequindox is rapidly metabolized to numerous metabolites following administration to animals. There have, however, been few reports describing the cytotoxicity of mequindox metabolites. In this study, HepG2 cells were treated with mequindox (0, 2, 10, 50 or 100 μg/ml) or its major metabolites (0, 40, 100, 250 or 500 μg/ml) for 24h. Mice were administrated with mequindox (0, 50, 200 or 500 mg/kg.bw) for five days. DNA damage in the HepG2 cells and mouse hepatocytes was then assessed using an SCGE assay. The cell cycle of the HepG2 cells was also determined by flow cytometry. Mequindox was found to induce cell cycle arrest to the G2/M phase and cause dose-dependent DNA damage in HepG2 cells in vitro and in murine hepatocytes in vivo. Compared with mequindox, the major metabolites had much smaller effects on the cell cycle and caused much less DNA damage in HepG2 cells. And the results indicated that the process of metabolites formed by reduction of the MEQ acetyl group or reduction of the N → O groups could contribute to DNA damage in murine hepatocytes in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Nickel chloride (NiCl2) in hepatic toxicity: apoptosis, G2/M cell cycle arrest and inflammatory response

PubMed Central

Guo, Hongrui; Cui, Hengmin; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Zhao, Ling; Chen, Kejie; Deng, Jie

2016-01-01

Up to now, the precise mechanism of Ni toxicology is still indistinct. Our aim was to test the apoptosis, cell cycle arrest and inflammatory response mechanism induced by NiCl2 in the liver of broiler chickens. NiCl2 significantly increased hepatic apoptosis. NiCl2 activated mitochondria-mediated apoptotic pathway by decreasing Bcl-2, Bcl-xL, Mcl-1, and increasing Bax, Bak, caspase-3, caspase-9 and PARP mRNA expression. In the Fas-mediated apoptotic pathway, mRNA expression levels of Fas, FasL, caspase-8 were increased. Also, NiCl2 induced ER stress apoptotic pathway by increasing GRP78 and GRP94 mRNA expressions. The ER stress was activated through PERK, IRE1 and ATF6 pathways, which were characterized by increasing eIF2α, ATF4, IRE1, XBP1 and ATF6 mRNA expressions. And, NiCl2 arrested G2/M phase cell cycle by increasing p53, p21 and decreasing cdc2, cyclin B mRNA expressions. Simultaneously, NiCl2 increased TNF-α, IL-1β, IL-6, IL-8 mRNA expressions through NF-κB activation. In conclusion, NiCl2 induces apoptosis through mitochondria, Fas and ER stress-mediated apoptotic pathways and causes cell cycle G2/M phase arrest via p53-dependent pathway and generates inflammatory response by activating NF-κB pathway. PMID:27824316

Activation of GPR30 inhibits growth of prostate cancer cells via sustained activation of Erk1/2, c-jun/c-fos-dependent upregulation of p21, and induction of G2 cell-cycle arrest

PubMed Central

Chan, Queeny K.Y.; Lam, Hung-Ming; Ng, Chi-Fai; Lee, Amy Y.Y.; Chan, Eddie S.Y.; Ng, Ho-Keung; Ho, Shuk-Mei; Lau, Kin-Mang

2010-01-01

G protein–coupled receptor 30 (GPR30) exhibits estrogen-binding affinity and mediates nongenomic signaling of estrogen to regulate cell growth. We here demonstrated for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited growth of androgen-dependent and -independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1-induced cell-cycle arrest at the G2 phase and reduced the expression of G2-checkpoint regulators (cyclin A2, cyclin B1, cdc25c, and cdc2) and the phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With the extensive use of siRNA knockdown experiments and the MEK inhibitor PD98059 in the present study, we dissected the mechanism underlying G-1–induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by a sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G2 phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa. PMID:20203690

Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin

2014-03-28

Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined themore » impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.« less

Immunohistochemical estimation of cell cycle phase in laryngeal neoplasia

PubMed Central

Chatrath, P; Scott, I S; Morris, L S; Davies, R J; Bird, K; Vowler, S L; Coleman, N

2006-01-01

We previously developed an immunohistochemical method for estimating cell cycle state and phase in tissue samples, including biopsies that are too small for flow cytometry. We have used our technique to examine whether primary abnormalities of the cell cycle exist in laryngeal neoplasia. Antibodies against the markers of cell cycle entry, minichromosome maintenance protein-2 (Mcm-2) and Ki67, and putative markers of cell cycle phase, cyclin D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis) were applied to paraffin-embedded sections of normal larynx (n=8), laryngeal dysplasia (n=10) and laryngeal squamous cell carcinoma (n=10). Cells expressing each marker were determined as a percentage of total cells, termed the labelling index (LI), and as a percentage of Mcm-2-positive cells, termed the labelling fraction (LF). The frequency of coexpression of each putative phase marker was investigated by confocal microscopy. There was a correlation between Mcm-2 and Ki67 LIs (ρ=0.93) but Mcm-2 LIs were consistently higher. All cells expressing a phase marker coexpressed Mcm-2, whereas Ki67 was not expressed in a proportion of these cells. The putative phase markers showed little coexpression. Labelling index values increased on progression from normal larynx through laryngeal dysplasia to squamous cell carcinoma for Mcm-2 (P=0.001), Ki67 (P=0.0002), cyclin D1 (P=0.015), cyclin A (P=0.0001) and cyclin B1 (P=0.0004). There was no evidence of an increase in the LF for any phase marker. Immunohistochemistry can be used to estimate cell cycle state and phase in laryngeal biopsies. Our data argues against primary cell cycle phase abnormalities in laryngeal neoplasia. PMID:16832409

Cytotoxicity assessments of Portulaca oleracea and Petroselinum sativum seed extracts on human hepatocellular carcinoma cells (HepG2).

PubMed

Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2014-01-01

The Pharmacological potential, such as antioxidant, anti-inflammatory, and antibacterial activities of Portulaca oleracea (PO) and Petroselinum sativum (PS) extracts are well known. However, the preventive properties against hepatocellular carcinoma cells have not been explored so far. Therefore, the present investigation was designed to study the anticancer activity of seed extracts of PO and PS on the human hepatocellular carcinoma cells (HepG2). The HepG2 cells were exposed with 5-500 μg/ml of PO and PS for 24 h. After the exposure, cell viability by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay, and cellular morphology by phase contrast inverted microscope were studied. The results showed that PO and PS extracts significantly reduced the cell viability of HepG2 in a concentration dependent manner. The cell viability was recorded to be 67%, 31%, 21%, and 17% at 50, 100, 250, and 500 μg/ml of PO, respectively by MTT assay and 91%, 62%, 27%, and 18% at 50, 100, 250, and 500 μg/ml of PO, respectively by NRU assay. PS exposed HepG2 cells with 100 μg/ml and higher concentrations were also found to be cytotoxic. The decrease in the cell viability at 100, 250, and 500 μg/ml of PS was recorded as 70%, 33%, and 15% by MTT assay and 63%, 29%, and 17%, respectively by NRU assay. Results also showed that PO and PS exposed cells reduced the normal morphology and adhesion capacity of HepG2 cells. HepG2 cells exposed with 50 μg/ml and higher concentrations of PO and PS lost their typical morphology, become smaller in size, and appeared in rounded bodies. Our results demonstrated preliminary screening of anticancer activity of Portulaca oleracea and Petroselinum sativum extracts against HepG2 cells, which can be further used for the development of a potential therapeutic anticancer agent.

Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

PubMed

Lee, Min Ho; Cho, Yoonjung; Jung, Byung Chul; Kim, Sung Hoon; Kang, Yeo Wool; Pan, Cheol-Ho; Rhee, Ki-Jong; Kim, Yoon Suk

2015-08-14

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

PubMed

Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai-Kei; Zhang, Wei

2015-07-26

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.

[6]-Gingerol enhances the radiosensitivity of gastric cancer via G2/M phase arrest and apoptosis induction.

PubMed

Luo, Youjun; Chen, Xue; Luo, Lumeng; Zhang, Qi; Gao, Caixia; Zhuang, Xibing; Yuan, Sujuan; Qiao, Tiankui

2018-05-01

Ionizing radiation (IR) is the main modality for locoregional control of unresectable gastric cancer (GC). [6]-Gingerol is an active major phenolic compound isolated from ginger (Zingiber officinale Roscoe), and it has been demonstrated to possess antitumor activity in previous studies. In the present study, we aimed to evaluate the potential activity of [6]-gingerol as a radiosensitizer and to further explore the underlying mechanism. A CCK-8 assay revealed that [6]-gingerol inhibited the cell viability of HGC-27 cells in a dose-dependent manner (P<0.05). Colony formation assay indicated that pretreatment of [6]-gingerol prior to IR decreased the clonogenic survival of HGC-27 cells. Notably, the combination of [6]-gingerol with IR enhanced IR-induced cell cycle arrest at the G2/M phase compared with IR alone (41.3% in IR alone vs. 53.5% in [6]-gingerol+IR; P=0.006), and increased IR-induced apoptosis compared with IR alone (9.6% in IR alone group vs. 15.1% in [6]-gingerol+IR; P=0.07). DAPI staining detected the apoptotic nuclear morphological changes in the cells treated with [6]-gingerol and/or IR. Furthermore, western blotting and qRT-PCR revealed that [6]-gingerol pretreatment following IR downregulated the protein expression of cyclin B1, cyclin A2, CDC2 and cyclin D1, upregulated the mRNA expression of p27, and induced active caspase-9, active caspase-3 and cytochrome c. In conclusion, the present study demonstrated that [6]-gingerol enhanced radiosensitivity of GC cells, and that the mechanisms involved at least G2/M phase arrest and apoptosis induction.

Inhibition of Rac1 activity induces G1/S phase arrest through the GSK3/cyclin D1 pathway in human cancer cells.

PubMed

Liu, Linna; Zhang, Hongmei; Shi, Lei; Zhang, Wenjuan; Yuan, Juanli; Chen, Xiang; Liu, Juanjuan; Zhang, Yan; Wang, Zhipeng

2014-10-01

Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

NASA Astrophysics Data System (ADS)

Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M-phase accumulation and apoptosis in multiple myeloma cells.

PubMed

Strömberg, Thomas; Ekman, Simon; Girnita, Leonard; Dimberg, Lina Y; Larsson, Olle; Axelson, Magnus; Lennartsson, Johan; Hellman, Ulf; Carlson, Kristina; Osterborg, Anders; Vanderkerken, Karin; Nilsson, Kenneth; Jernberg-Wiklund, Helena

2006-01-15

Emerging evidence suggests the insulin-like growth factor-1 receptor (IGF-1R) to be an important mediator of tumor-cell survival and resistance to cytotoxic therapy in multiple myeloma (MM). Recently, members of the cyclolignan family have been shown to selectively inhibit the receptor tyrosine kinase (RTK) activity of the IGF-1R beta-chain. The effects of the cyclolignan picropodophyllin (PPP) were studied in vitro using a panel of 13 MM cell lines and freshly purified tumor cells from 10 patients with MM. PPP clearly inhibited growth in all MM cell lines and primary MM samples cultured in the presence or absence of bone marrow stromal cells. PPP induced a profound accumulation of cells in the G(2)/M-phase and an increased apoptosis. Importantly, IGF-1, IGF-2, insulin, or IL-6 did not reduce the inhibitory effects of PPP. As demonstrated by in vitro kinase assays, PPP down-regulated the IGF-1 RTK activity without inhibiting the insulin RTK activity. This conferred decreased phosphorylation of Erk1/2 and reduced cyclin dependent kinase (CDK1) activity. In addition, the expression of mcl-1 and survivin was reduced. Taken together, we suggest that interfering with the IGF-1 RTK by using the cyclolignan PPP offers a novel and selective therapeutic strategy for MM.

Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com; Cheah, Yew-Hoong; Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur

Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action ofmore » xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.« less

Palmitic acid-induced neuron cell cycle G2/M arrest and endoplasmic reticular stress through protein palmitoylation in SH-SY5Y human neuroblastoma cells.

PubMed

Hsiao, Yung-Hsuan; Lin, Ching-I; Liao, Hsiang; Chen, Yue-Hua; Lin, Shyh-Hsiang

2014-11-13

Obesity-related neurodegenerative diseases are associated with elevated saturated fatty acids (SFAs) in the brain. An increase in SFAs, especially palmitic acid (PA), triggers neuron cell apoptosis, causing cognitive function to deteriorate. In the present study, we focused on the specific mechanism by which PA triggers SH-SY5Y neuron cell apoptosis. We found that PA induces significant neuron cell cycle arrest in the G2/M phase in SH-SY5Y cells. Our data further showed that G2/M arrest is involved in elevation of endoplasmic reticular (ER) stress according to an increase in p-eukaryotic translation inhibition factor 2α, an ER stress marker. Chronic exposure to PA also accelerates beta-amyloid accumulation, a pathological characteristic of Alzheimer's disease. Interestingly, SFA-induced ER stress, G2/M arrest and cell apoptosis were reversed by treatment with 2-bromopalmitate, a protein palmitoylation inhibitor. These findings suggest that protein palmitoylation plays a crucial role in SFA-induced neuron cell cycle G2/M arrest, ER stress and apoptosis; this provides a novel strategy for preventing SFA-induced neuron cell dysfunction.

Alkali-soluble polysaccharide, isolated from Lentinus edodes, induces apoptosis and G2/M cell cycle arrest in H22 cells through microtubule depolymerization.

PubMed

You, Ru-Xu; Liu, Jin-Yu; Li, Shi-Jun; Wang, Liu; Wang, Kai-Ping; Zhang, Yu

2014-12-01

The aim of the study was to evaluate the pro-apoptotic effects of polysaccharides derived from Lentinus edodes and further elucidated the mechanisms of this action. Our results demonstrated that marked morphological changes of apoptosis were observed after treatment of L. edodes polysaccharides [Lentinan (LTN)]. Moreover, LTN-induced cell apoptosis was characterized by a rapid stimulation of reactive oxygen species production, the loss of mitochondrial membrane potential and an increase in intracellular concentration of Ca(2+) . In addition, the results of the haematoxylin and eosin and TUNEL assay further confirmed that LTN-induced apoptosis in vivo. Furthermore, flow cytometry analysis showed that LTN could arrest the cell cycle at G2/M phase, and immunofluorescence showed LTN caused disruption of microtubule. These results suggest that disruption of cellular microtubule network, arrest of the cell cycle at G2/M phase and induction of apoptosis may be one of the possible mechanisms of anti-tumour effect of LTN. Copyright © 2014 John Wiley & Sons, Ltd.

G0-G1 Transition and the Restriction Point in Pancreatic β-Cells In Vivo

PubMed Central

Hija, Ayat; Salpeter, Seth; Klochendler, Agnes; Grimsby, Joseph; Brandeis, Michael; Glaser, Benjamin; Dor, Yuval

2014-01-01

Most of our knowledge on cell kinetics stems from in vitro studies of continuously dividing cells. In this study, we determine in vivo cell-cycle parameters of pancreatic β-cells, a largely quiescent population, using drugs that mimic or prevent glucose-induced replication of β-cells in mice. Quiescent β-cells exposed to a mitogenic glucose stimulation require 8 h to enter the G1 phase of the cell cycle, and this time is prolonged in older age. The duration of G1, S, and G2/M is ∼5, 8, and 6 h, respectively. We further provide the first in vivo demonstration of the restriction point at the G0-G1 transition, discovered by Arthur Pardee 40 years ago. The findings may have pharmacodynamic implications in the design of regenerative therapies aimed at increasing β-cell replication and mass in patients with diabetes. PMID:24130333

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway.

PubMed

Yang, Yang; Li, Xi; Sun, Qinwei; He, Bin; Jia, Yimin; Cai, Demin; Zhao, Ruqian

2016-10-01

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

PubMed

Tsai, Tsung-Chang; Huang, Hui-Pei; Chang, Kai-Ting; Wang, Chau-Jong; Chang, Yun-Ching

2017-04-01

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL -1 ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017. © 2016 Wiley Periodicals, Inc.

Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

PubMed

Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

2010-04-01

Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins

PubMed Central

LV, TING-ZHUO; WANG, GUANG-SHUN

2015-01-01

Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera. This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins. PMID:26170956

Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins.

PubMed

Lv, Ting-Zhuo; Wang, Guang-Shun

2015-07-01

Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera . This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins.

Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cells.

PubMed

Alja, Štraser; Filipič, Metka; Novak, Matjaž; Žegura, Bojana

2013-08-21

The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1-0.5 µg/mL, 24-96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.

Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

PubMed

de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

2015-10-02

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects

The impact of miR-34a on protein output in hepatocellular carcinoma HepG2 cells.

PubMed

Cheng, Jun; Zhou, Lin; Xie, Qin-Fen; Xie, Hai-Yang; Wei, Xu-Yong; Gao, Feng; Xing, Chun-Yang; Xu, Xiao; Li, Lan-Juan; Zheng, Shu-Sen

2010-04-01

MicroRNAs are small non-coding RNA molecules that play essential roles in biological processes ranging from cell cycle to cell migration and invasion. Accumulating evidence suggests that miR-34a, as a key mediator of p53 tumor suppression, is aberrantly expressed in human cancers. In the present study, we aimed to explore the precise biological role of miR-34a and the global protein changes in HCC cell line HepG2 cells transiently transfected with miR-34a. Transfection of miR-34a into HepG2 cells caused suppression of cell proliferation, inhibition of cell migration and invasion. It also induced an accumulation of HepG2 cells in G1 phase. Among 116 protein spots with differential expression separated by 2-DE method, 34 proteins were successfully identified by MALDI-TOF/TOF analysis. Of these, 15 downregulated proteins may be downstream targets of miR-34a. Bioinformatics analysis produced a protein-protein interaction network, which revealed that the p53 signaling pathway and cell cycle pathway were two major hubs containing most of the proteins regulated by miR-34a. Cytoskeletal proteins such as LMNA, GFAP, MACF1, ALDH2, and LOC100129335 are potential targets of miR-34a. In conclusion, abrogation of miR-34a function could cause downstream molecules to switch on or off, leading to HCC development.

Inhibition of Aurora A Kinase by Alisertib Induces Autophagy and Cell Cycle Arrest and Increases Chemosensitivity in Human Hepatocellular Carcinoma HepG2 Cells.

PubMed

Zhu, Qiaohua; Yu, Xinfa; Zhou, Zhi-Wei; Zhou, Chengyu; Chen, Xiao-Wu; Zhou, Shu-Feng

2017-01-01

Aurora A kinase represent a feasible target in cancer therapy. To evaluate the proteomic response of human liver carcinoma cells to alisertib (ALS) and identify the molecular targets of ALS, we examined the effects of ALS on the proliferation, cell cycle, autophagy, apoptosis, and chemosensitivity in HepG2 cells. The stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomic study was performed to evaluate the proteomic response to ALS. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. Our SILAC proteomic study showed that ALS regulated the expression of 914 proteins, with 407 molecules being up-regulated and 507 molecules being down-regulated in HepG2 cells. Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 146 and 32 signaling pathways were regulated by ALS, respectively, which were associated with cell survival, programmed cell death, and nutrition-energy metabolism. Subsequently, the verification experiments showed that ALS remarkably arrested HepG2 cells in G2/M phase and led to an accumulation of aneuploidy via regulating the expression of key cell cycle regulators. ALS induced a marked autophagy in a concentration- and time-dependent manner via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Autophagy inhibition promoted the pro-apoptotic effect of ALS, indicating a cyto-protective role of ALS-induced autophagy. ALS increased the chemosensitivity of HepG2 cells to cisplatin and doxorubicin. Taken together, ALS induces autophagy and cell cycle arrest in HepG2 cells via PI3K/Akt/mTOR-mediated pathway. Autophagy inhibition may promote the anticancer effect of ALS and sensitize the chemotherapy in HepG2 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Determination of cell cycle phases in live B16 melanoma cells using IRMS.

PubMed

Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

2013-07-21

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

Estrogen and progesterone promote breast cancer cell proliferation by inducing cyclin G1 expression.

PubMed

Tian, J-M; Ran, B; Zhang, C-L; Yan, D-M; Li, X-H

2018-01-23

Breast cancer is the most common cause of cancer among women in most countries (WHO). Ovarian hormone disorder is thought to be associated with breast tumorigenesis. The present study investigated the effects of estrogen and progesterone administration on cell proliferation and underlying mechanisms in breast cancer MCF-7 cells. It was found that a single administration of estradiol (E2) or progesterone increased MCF-7 cell viability in a dose-dependent manner and promoted cell cycle progression by increasing the percentage of cells in the G2/M phase. A combination of E2 and progesterone led to a stronger effect than single treatment. Moreover, cyclin G1 was up-regulated by E2 and/or progesterone in MCF-7 cells. After knockdown of cyclin G1 in MCF-7 cells using a specific shRNA, estradiol- and progesterone-mediated cell viability and clonogenic ability were significantly limited. Additionally, estradiol- and progesterone-promoted cell accumulation in the G2/M phase was reversed after knockdown of cyclin G1. These data indicated that estrogen and progesterone promoted breast cancer cell proliferation by inducing the expression of cyclin G1. Our data indicated that novel therapeutics against cyclin G1 are promising for the treatment of estrogen- and progesterone-mediated breast cancer progression.

Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line.

PubMed

Yu, Jian-Qing; Yin, Yan; Lei, Jia-Chuan; Zhang, Xiu-Qiao; Chen, Wei; Ding, Cheng-Li; Wu, Shan; He, Xiao-Yu; Liu, Yan-Wen; Zou, Guo-Lin

2012-02-01

Dianthus superbus L. is commonly used as a traditional Chinese medicine. We recently showed that ethyl acetate fraction (EE-DS) from ethanol extract of D. superbus exhibited the strongest antioxidant and cytotoxic activities. In this study, we examined apoptosis of HepG2 cells induced by EE-DS, and the mechanism underlying apoptosis was also investigated. Treatment of HepG2 cells with EE-DS (20-80 μg/ml) for 48 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a large number of apoptotic bodies containing nuclear fragments were observed in cells treated with 80 μg/ml of EE-DS for 24 h by using Hoechst 33258 staining. These data show that EE-DS can induce apoptosis of HepG2 cells. Immunoblot analysis showed that EE-DS significantly suppressed the expressions of Bcl-2 and NF-κB. Treatment of cells with EE-DS (80 μg/ml) for 48 h resulted in significant increase of cytochrome c in the cytosol, which indicated cytochrome c release from mitochondria. Activation of caspase-9 and -3 were also determined when the cells treated with EE-DS. The results suggest that apoptosis of HepG2 cells induced by EE-DS could be through the mitochondrial intrinsic pathway. High performance liquid chromatography (HPLC) data showed that the composition of EE-DS is complicated. Further studies are needed to find the effective constituents of EE-DS. Copyright © 2011 Elsevier Ltd. All rights reserved.

Infrared spectroscopic studies of myeloid leukemia (ML-1) cells at different phases of the cell cycle

NASA Astrophysics Data System (ADS)

Boydston-White, Susie; Diem, Max

1999-06-01

Advances in infrared spectroscopic methodology permit excellent infrared spectra to be collected from objects as small as single human cells. These advances have lead to an increased interest of the use of infrared spectroscopy as a medical diagnostic tool. Infrared spectra of myeloid leukemia (ML-1) cells are reported for cells derived from an asynchronous, exponentially-growing culture, as well as for cells that were fractionated according to their stage within the cell division cycle. The observed results suggest that the cells' DNA is detectable by infrared spectroscopy mainly when the cell is in the S phase, during the replication of DNA. In the G1 and G2 phases, the DNA is so tightly packed in the nucleus that it appears opaque to infrared radiation. Consequently, the nucleic acid spectral contributions in the G1 and G2 phases would be mostly that of cytoplasmic RNA. These results suggest that infrared spectral changes observed earlier between normal and abnormal cells may have been due to different distributions of cells within the stages of the cell division cycle.

Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

PubMed Central

Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

1987-01-01

One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978

Inhibition of HBV Replication in HepG2.2.15 Cells by Human Peripheral Blood Mononuclear Cell-Derived Dendritic Cells.

PubMed

Liu, Tao; Song, Hong-Li; Zheng, Wei-Ping; Shen, Zhong-Yang

2015-01-01

Anti-HBV therapy is essential for patients awaiting liver transplantation. This study aimed to explore the effects of dendritic cells (DCs) derived from the peripheral blood of hepatitis B patients on the replication of HBV in vivo and to evaluate the biosafety of DCs in clinical therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from HBV-infected patients and maturation-promoting factors and both HBsAg and HBcAg were used to induce DC maturation. Mature DCs and lymphocytes were co-cultured with human hepatocyte cell HL-7702 or HBV-producing human hepatocellular carcinoma cell HepG2.2.15. We found that mature lymphocytes exposed to DCs in vitro did not influence morphology or activities of HL-7702 and HepG2.2.15 cells. Liver function indexes and endotoxin levels in the cell supernatants did not change in these co-cultures. Additionally, supernatant and intracellular HBV DNA levels were reduced when HepG2.2.15 cells were co-cultured with mature lymphocytes that had been cultured with DCs, and HBV covalently closed circular DNA (cccDNA) levels in HepG2.2.15 cells also decreased. Importantly, DC-mediated immunotherapy had no mutagenic effect on HBV genomic DNA by gene sequencing of the P, S, X, and C regions of HBV genomic DNA. We conclude that PBMC-derived DCs from HBV-infected patients act on autologous lymphocytes to suppress HBV replication and these DC clusters showed favorable biosafety. © 2015 by the Association of Clinical Scientists, Inc.

Novel ferrocenyl derivatives exert anti-cancer effect in human lung cancer cells in vitro via inducing G1-phase arrest and senescence

PubMed Central

Li, Ying; Ma, Han-lin; Han, Lei; Liu, Wei-yong; Zhao, Bao-xiang; Zhang, Shang-li; Miao, Jun-ying

2013-01-01

Aim: To investigate the effects of 7 novel 1-ferrocenyl-2-(5-phenyl-1H-1,2,4-triazol-3-ylthio) ethanone derivatives on human lung cancer cells in vitro and to determine the mechanisms of action. Methods: A549 human lung cancer cells were examined. Cell viability was analyzed with MTT assay. Cell apoptosis and senescence were examined using Hoechst 33258 and senescence-associated-β-galactosidase (SA-β-gal) staining, respectively. LDH release was measured using a detection kit. Cell cycle was analyzed using a flow cytometer. Intracellular ROS level was measured with the 2′,7′-dichlorodihydrofluorescein probe. Phosphorylation of p38 was determined using Western blot. Results: Compounds 5b, 5d, and 5e (40 and 80 μmol/L) caused significant decrease of A549 cell viability, while other 4 compounds had no effect on the cells. Compounds 5b, 5d, and 5e (80 μmol/L) induced G1-phase arrest (increased the G1 population by 22.6%, 24.23%, and 26.53%, respectively), and markedly increased SA-β-gal-positive cells. However, the compounds did not cause nuclear DNA fragmentation and chromatin condensation in A549 cells. Nor did they affect the release of LDH from the cells. The compounds significantly elevated the intracellular ROS level, decreased the mitochondrial membrane potential, and increased p38 phosphorylation in the cells. In the presence of the antioxidant and free radical scavenger N-acetyl-L-cysteine (10 mmol/L), above effects of compounds 5b, 5d, and 5e were abolished. Conclusion: The compounds 5b, 5d, and 5e cause neither apoptosis nor necrosis of A549 cells, but exert anti-cancer effect via inducing G1-phase arrest and senescence through ROS/p38 MAP-kinase pathway. PMID:23645009

6-Gingerol Inhibits Growth of Colon Cancer Cell LoVo via Induction of G2/M Arrest

PubMed Central

Lin, Ching-Bin; Lin, Chun-Che; Tsay, Gregory J.

2012-01-01

6-Gingerol, a natural component of ginger, has been widely reported to possess antiinflammatory and antitumorigenic activities. Despite its potential efficacy against cancer, the anti-tumor mechanisms of 6-gingerol are complicated and remain sketchy. In the present study, we aimed to investigate the anti-tumor effects of 6-gingerol on colon cancer cells. Our results revealed that 6-gingerol treatment significantly reduced the cell viability of human colon cancer cell, LoVo, in a dose-dependent manner. Further flow cytometric analysis showed that 6-gingerol induced significant G2/M phase arrest and had slight influence on sub-G1 phase in LoVo cells. Therefore, levels of cyclins, cyclin-dependent kinases (CDKs), and their regulatory proteins involved in S-G2/M transition were investigated. Our findings revealed that levels of cyclin A, cyclin B1, and CDK1 were diminished; in contrast, levels of the negative cell cycle regulators p27Kip1 and p21Cip1 were increased in response to 6-gingerol treatment. In addition, 6-gingerol treatment elevated intracellular reactive oxygen species (ROS) and phosphorylation level of p53. These findings indicate that exposure of 6-gingerol may induce intracellular ROS and upregulate p53, p27Kip1, and p21Cip1 levels leading to consequent decrease of CDK1, cyclin A, and cyclin B1 as result of cell cycle arrest in LoVo cells. It would be suggested that 6-gingerol should be beneficial to treatment of colon cancer. PMID:22719783

6-Gingerol Inhibits Growth of Colon Cancer Cell LoVo via Induction of G2/M Arrest.

PubMed

Lin, Ching-Bin; Lin, Chun-Che; Tsay, Gregory J

2012-01-01

6-Gingerol, a natural component of ginger, has been widely reported to possess antiinflammatory and antitumorigenic activities. Despite its potential efficacy against cancer, the anti-tumor mechanisms of 6-gingerol are complicated and remain sketchy. In the present study, we aimed to investigate the anti-tumor effects of 6-gingerol on colon cancer cells. Our results revealed that 6-gingerol treatment significantly reduced the cell viability of human colon cancer cell, LoVo, in a dose-dependent manner. Further flow cytometric analysis showed that 6-gingerol induced significant G2/M phase arrest and had slight influence on sub-G1 phase in LoVo cells. Therefore, levels of cyclins, cyclin-dependent kinases (CDKs), and their regulatory proteins involved in S-G2/M transition were investigated. Our findings revealed that levels of cyclin A, cyclin B1, and CDK1 were diminished; in contrast, levels of the negative cell cycle regulators p27(Kip1) and p21(Cip1) were increased in response to 6-gingerol treatment. In addition, 6-gingerol treatment elevated intracellular reactive oxygen species (ROS) and phosphorylation level of p53. These findings indicate that exposure of 6-gingerol may induce intracellular ROS and upregulate p53, p27(Kip1), and p21(Cip1) levels leading to consequent decrease of CDK1, cyclin A, and cyclin B1 as result of cell cycle arrest in LoVo cells. It would be suggested that 6-gingerol should be beneficial to treatment of colon cancer.

TGFβ lengthens the G1 phase of stem cells in aged mouse brain.

PubMed

Daynac, Mathieu; Pineda, Jose R; Chicheportiche, Alexandra; Gauthier, Laurent R; Morizur, Lise; Boussin, François D; Mouthon, Marc-André

2014-12-01

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique, we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition, we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells, but not in transit-amplifying cells, and directly impacts on neurogenesis. Finally, we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. © 2014 AlphaMed Press.

Flipping the Switch from G1 to S Phase with E3 Ubiquitin Ligases

PubMed Central

Rizzardi, Lindsay F.

2012-01-01

The cell cycle ensures genome maintenance by coordinating the processes of DNA replication and chromosome segregation. Of particular importance is the irreversible transition from the G1 phase of the cell cycle to S phase. This transition marks the switch from preparing chromosomes for replication (“origin licensing”) to active DNA synthesis (“origin firing”). Ubiquitin-mediated proteolysis is essential for restricting DNA replication to only once per cell cycle and is the major mechanism regulating the G1 to S phase transition. Although some changes in protein levels are attributable to regulated mRNA abundance, protein degradation elicits very rapid changes in protein abundance and is critical for the sharp and irreversible transition from one cell cycle stage to the next. Not surprisingly, regulation of the G1-to-S phase transition is perturbed in most cancer cells, and deregulation of key molecular events in G1 and S phase drives not only cell proliferation but also genome instability. In this review we focus on the mechanisms by which E3 ubiquitin ligases control the irreversible transition from G1 to S phase in mammalian cells. PMID:23634252

Umbilical cord-derived mesenchymal stem cells inhibit growth and promote apoptosis of HepG2 cells.

PubMed

Tang, Ying-Mei; Bao, Wei-Min; Yang, Jin-Hui; Ma, Lin-Kun; Yang, Jing; Xu, Ying; Yang, Li-Hong; Sha, Feng; Xu, Zhi-Yuan; Wu, Hua-Mei; Zhou, Wei; Li, Yan; Li, Yu-Hua

2016-09-01

Hepatocellular carcinoma is the fifth most common type of cancer worldwide and remains difficult to treat. The aim of this study was to investigate the effects of mesenchymal stem cells (MSCs) derived from the umbilical cord (UC‑MSCs) on HepG2 hepatocellular carcinoma cells. UC‑MSCs were co‑cultured with HepG2 cells and biomarkers of UC‑MSCs were analyzed by flow cytometry. mRNA and protein expression of genes were determined by reverse transcription‑polymerase chain reaction and flow cytometry, respectively. Passage three and seven UC‑MSCs expressed CD29, CD44, CD90 and CD105, whereas CD34 and CD45 were absent on these cells. Co‑culture with UC‑MSCs inhibited proliferation and promoted apoptosis of HepG2 cells in a time‑dependent manner. The initial seeding density of UC‑MSCs also influenced the proliferation and apoptosis of HepG2 cells, with an increased number of UC‑MSCs causing enhanced proliferation inhibition and cell apoptosis. Co‑culture with UC‑MSCs downregulated mRNA and protein expression of α‑fetoprotein (AFP), Bcl‑2 and Survivin in HepG2 cells. Thus, UC‑MSCs may inhibit growth and promote apoptosis of HepG2 cells through downregulation of AFP, Bcl‑2 and Survivin. US-MSCs may be used as a novel therapy for treating hepatocellular carcinoma in the future.

Lipotoxicity in HepG2 cells triggered by free fatty acids

PubMed Central

Yao, Hong-Rui; Liu, Jun; Plumeri, Daniel; Cao, Yong-Bing; He, Ting; Lin, Ling; Li, Yu; Jiang, Yuan-Ying; Li, Ji; Shang, Jing

2011-01-01

The goal of this study was to investigate the lipid accumulation and lipotoxicity of free fatty acids (FFAs) induced in HepG2 cells. HepG2 cells were co-incubated with various concentrations of FFAs for 24h and the intracellular lipid contents were observed by Oil Red O and Nile Red staining methods. The lipotoxicity of HepG2 cells were then detected by Hoechest 33342/PI, Annexin V-FITC/PI double-staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) experiment tests. The experiments showed a lipid accumulation and lipotoxicity by increasing FFA concentration gradients. Through cell morphological observation and quantitative analysis, FFAs have shown to increase in a dose-dependent manner compared with the control group. The data collected from hoechst 33342/PI, annexin V-FITC/PI double staining and also MTT experiments showed that cell apoptosis and necrosis significantly increased with increasing FFA concentrations. Apoptosis was not obvious in the 1 mM FFAs-treated group compared to the other two groups. In a certain concentration range, FFAs induced intracellular lipid accumulation and lipotoxicity of HepG2 cells in a dose-dependent manner. PMID:21654881

Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14.

PubMed

Nanbo, A; Nishimura, H; Muta, T; Nagasawa, S

1999-02-01

Lipopolysaccharide (LPS)-binding protein (LBP), an opsonin for activation of macrophages by bacterial LPS, is synthesized in hepatocytes and is known to be an acute phase protein. Recently, cytokine-induced production of LBP was reported to increase 10-fold in hepatocytes isolated from LPS-treated rats, compared with those from normal rats. However, the mechanism by which the LPS treatment enhances the effect of cytokines remains to be clarified. In the present study, we examined whether LPS alone or an LPS/LBP complex directly stimulates the hepatocytes, leading to acceleration of the cytokine-induced LBP production. HepG2 cells (a human hepatoma cell line) were shown to express CD14, a glycosylphosphatidylinositol-anchored LPS receptor, by both RT/PCR and flow cytometric analyses. An LPS/LBP complex was an effective stimulator for LBP and CD14 production in HepG2 cells, but stimulation of the cells with either LPS or LBP alone did not significantly accelerate the production of these proteins. The findings were confirmed by semiquantitative RT/PCR analysis of mRNA levels of LBP and CD14 in HepG2 cells after stimulation with LPS alone and an LPS/LBP complex. In addition, two monoclonal antibodies (mAbs) to CD14 (3C10 and MEM-18) inhibited LPS/LBP-induced cellular responses of HepG2 cells. Furthermore, prestimulation of HepG2 cells with LPS/LBP augmented cytokine-induced production and gene expression of LBP and CD14. All these findings suggest that an LPS/LBP complex, but not free LPS, stimulates HepG2 cells via CD14 leading to increased basal and cytokine-induced LBP and CD14 production.

IgG2 deficiency in sickle cell anaemia.

PubMed

Natta, C L; Outschoorn, I M

1984-08-01

8 patients with known sickle cell anaemia were studied immunologically. The concentrations of the main immunoglobulin classes, IgG and IgA, were significantly higher than the levels in 11 normal age- and sex-matched black subjects (P less than 0.01). IgM levels were not significantly different in the two groups. There was a heterogeneity in the interaction of the IgG subclasses with Protein A, with low levels of IgG2. The IgG2:IgG1 ratios varied from 1:3.8 to 1:6 (normals 1:3). In 4 patients the absolute levels of IgG2 as measured by radial immunodiffusion were lower than normal, thus confirming the chromatographic ratios. Since specific antibody is often restricted to a single subclass, the levels of IgG subclasses may be related to recurrent bacterial infections in these patients.

Selective cytotoxicity of PAMAM G5 core–PAMAM G2.5 shell tecto-dendrimers on melanoma cells

PubMed Central

Schilrreff, Priscila; Mundiña-Weilenmann, Cecilia; Romero, Eder Lilia; Morilla, Maria Jose

2012-01-01

Background The controlled introduction of covalent linkages between dendrimer building blocks leads to polymers of higher architectural order known as tecto-dendrimers. Because of the few simple steps involved in their synthesis, tecto-dendrimers could expand the portfolio of structures beyond commercial dendrimers, due to the absence of synthetic drawbacks (large number of reaction steps, excessive monomer loading, and lengthy chromatographic separations) and structural constraints of high-generation dendrimers (reduction of good monodispersity and ideal dendritic construction due to de Gennes dense-packing phenomenon). However, the biomedical uses of tecto-dendrimers remain unexplored. In this work, after synthesizing saturated shell core–shell tecto-dendrimers using amine-terminated polyamidoamine (PAMAM) generation 5 (G5) as core and carboxyl-terminated PAMAM G2.5 as shell (G5G2.5 tecto-dendrimers), we surveyed for the first time the main features of their interaction with epithelial cells. Methods Structural characterization of G5G2.5 was performed by polyacrylamide gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry, and microscopic techniques; their hydrodynamic size and Z-potential was also determined. Cellular uptake by human epidermal keratinocytes, colon adenocarcinoma, and epidermal melanoma (SK-Mel-28) cells was determined by flow cytometry. Cytotoxicity was determined by mitochondrial activity, lactate dehydrogenase release, glutathione depletion, and apoptosis/necrosis measurement. Results The resultant 60%–67% saturated shell, 87,000-dalton G5G2.5 (mean molecular weight) interacted with cells in a significantly different fashion in comparison to their building blocks and to its closest counterpart, PAMAM G6.5. After being actively taken up by epithelial cells, G5G2.5 caused cytotoxicity only on SK-Mel-28 cells, including depletion of intracellular glutathione and fast necrosis that was manifested above 5 μM G5

Transcription-dependent induction of G1 phase during the zebra fish midblastula transition.

PubMed

Zamir, E; Kam, Z; Yarden, A

1997-02-01

The early development of the zebra fish (Danio rerio) embryo is characterized by a series of rapid and synchronous cell cycles with no detectable transcription. This period is followed by the midblastula transition (MBT), during which the cell cycle gradually lengthens, cell synchrony is lost, and zygotic transcription is initially detected. In this work, we examined the changes in the pattern of the cell cycle during MBT in zebra fish and whether these changes are dependent on the initiation of zygotic transcription. To characterize the pattern of the early zebra fish cell cycles, the embryonic DNA content was determined by flow cytometric analysis. We found that G1 phase is below detection levels during the first 10 cleavages and can be initially detected at the onset of MBT. Inhibition of zygotic transcription, by microinjection of actinomycin D, abolished the appearance of G1 phase at MBT. Premature activation of zygotic transcription, by microinjection of nonspecific DNA, induced G1 phase before the onset of MBT, while coinjection of actinomycin D and nonspecific DNA abolished this early appearance of G1 phase. We therefore suggest that during the early development of the zebra fish embryo, G1 phase appears at the onset of MBT and that the activation of transcription at MBT is essential and sufficient for the G1-phase induction.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

1993-01-01

A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

1993-02-09

A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Anti-cancer Activity of Osmanthus matsumuranus Extract by Inducing G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma Hep G2 Cells

PubMed Central

Jin, Soojung; Park, Hyun-Jin; Oh, You Na; Kwon, Hyun Ju; Kim, Jeong-Hwan; Choi, Yung Hyun; Kim, Byung Woo

2015-01-01

Background: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). Methods: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. Results: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. Conclusions: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry. PMID:26734586

JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

PubMed

Liu, Zhengyun; Li, Guangmin; Gou, Ying; Xiao, Dongyan; Luo, Guo; Saavedra, Joseph E; Liu, Jie; Wang, Huan

2017-08-01

Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20μM) increased the expression of DNA damage-associated protein phosphorylation H 2 AX (γH 2 AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kito, Hiroaki; Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto; Yamamura, Hisao

2015-04-10

Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cyclemore » progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.« less

The Temporal Regulation of S Phase Proteins During G1

PubMed Central

Grant, Gavin D.; Cook, Jeanette G.

2018-01-01

Successful DNA replication requires intimate coordination with cell cycle progression. Prior to DNA replication initiation in S phase, a series of essential preparatory events in G1 phase ensures timely, complete, and precise genome duplication. Among the essential molecular processes are regulated transcriptional upregulation of genes that encode replication proteins, appropriate post-transcriptional control of replication factor abundance and activity, and the assembly of DNA-loaded protein complexes to license replication origins. In this chapter we describe these critical G1 events necessary for DNA replication and their regulation in the context of both cell cycle entry and cell cycle progression. PMID:29357066

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G{sub 2}/M arrest, and triggers apoptosis in human leukemia HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magalhães, Hemerson I.F.; Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, Paraíba; Wilke, Diego V.

2013-10-01

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC{sub 50} values in the nanomolar range. Cell cycle arrest in G{sub 2}/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation,more » loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G{sub 2}/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G{sub 2}/M phase of the cell cycle. • PHT induces caspase-dependent apoptosis.« less

Extremely Low-Frequency Electromagnetic Fields Cause G1 Phase Arrest through the Activation of the ATM-Chk2-p21 Pathway

PubMed Central

Huang, Chao-Ying; Chang, Cheng-Wei; Chen, Chaang-Ray; Chuang, Chun-Yu; Chiang, Chi-Shiun; Shu, Wun-Yi; Fan, Tai-Ching; Hsu, Ian C.

2014-01-01

In daily life, humans are exposed to the extremely low-frequency electromagnetic fields (ELF-EMFs) generated by electric appliances, and public concern is increasing regarding the biological effects of such exposure. Numerous studies have yielded inconsistent results regarding the biological effects of ELF-EMF exposure. Here we show that ELF-EMFs activate the ATM-Chk2-p21 pathway in HaCaT cells, inhibiting cell proliferation. To present well-founded results, we comprehensively evaluated the biological effects of ELF-EMFs at the transcriptional, protein, and cellular levels. Human HaCaT cells from an immortalized epidermal keratinocyte cell line were exposed to a 1.5 mT, 60 Hz ELF-EMF for 144 h. The ELF-EMF could cause G1 arrest and decrease colony formation. Protein expression experiments revealed that ELF-EMFs induced the activation of the ATM/Chk2 signaling cascades. In addition, the p21 protein, a regulator of cell cycle progression at G1 and G2/M, exhibited a higher level of expression in exposed HaCaT cells compared with the expression of sham-exposed cells. The ELF-EMF-induced G1 arrest was diminished when the CHK2 gene expression (which encodes checkpoint kinase 2; Chk2) was suppressed by specific small interfering RNA (siRNA). These findings indicate that ELF-EMFs activate the ATM-Chk2-p21 pathway in HaCaT cells, resulting in cell cycle arrest at the G1 phase. Based on the precise control of the ELF-EMF exposure and rigorous sham-exposure experiments, all transcriptional, protein, and cellular level experiments consistently supported the conclusion. This is the first study to confirm that a specific pathway is triggered by ELF-EMF exposure. PMID:25111195

Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells.

PubMed

Kito, Hiroaki; Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto; Ohya, Susumu; Asai, Kiyofumi; Imaizumi, Yuji

2015-04-10

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Bog bilberry (Vaccinium uliginosum L.) extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability.

PubMed

Liu, Jia; Zhang, Wei; Jing, Hao; Popovich, David G

2010-04-01

Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

PubMed

Haller, K; Ruckes, T; Schmitt, I; Saul, D; Derow, E; Grassmann, R

2000-11-01

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.

3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a novel combretastatin A-4 analog, induces G2/M arrest and apoptosis by disrupting tubulin polymerization in human cervical HeLa cells and fibrosarcoma HT-1080 cells.

PubMed

Zuo, Daiying; Guo, Dandan; Jiang, Xuewei; Guan, Qi; Qi, Huan; Xu, Jingwen; Li, Zengqiang; Yang, Fushan; Zhang, Weige; Wu, Yingliang

2015-02-05

Microtubule is a popular target for anticancer drugs. In this study, we describe the effect 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a newly synthesized analog of combretastatin A-4 (CA-4), showing a strong time- and dose-dependent anti-proliferative effect on human cervical cancer HeLa cells and human fibrosarcoma HT-1080 cells. We demonstrated that the growth inhibitory effects of G-1103 in HeLa and HT-1080 cells were associated with microtubule depolymerization and proved that G-1103 acted as microtubule destabilizing agent. Furthermore, cell cycle analysis revealed that G-1103 treatment resulted in cell cycle arrest at the G2/M phase in a time-dependent manner with subsequent apoptosis induction. Western blot analysis revealed that down-regulation of cdc25c and up-regulation of cyclin B1 was related with G2/M arrest in HeLa and HT-1080 cells treatment with G-1103. In addition, G-1103 induced HeLa cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8 expression, which indicated that G-1103 induced HeLa cell apoptosis was mainly associated with death receptor pathway. However, G-1103 induced HT-1080 cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8, Bax and cleaved caspase-9 expression and down-regulating anti-apoptotic protein Bcl-2 expression, which indicated that G-1103 induced HT-1080 cell apoptosis was associated with both mitochondrial and death receptor pathway. Taken together, all the data demonstrated that G-1103 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in HeLa and HT-1080 cells. Therefore, the novel compound G-1103 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

2012-07-01

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

Alteration of mitochondrial membrane potential by Spirulina platensis C-phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellular-carcinoma cell line HepG2.

PubMed

Roy, Karnati R; Arunasree, Kalle M; Reddy, Nishant P; Dheeraj, Bhavanasi; Reddy, Gorla Venkateswara; Reddanna, Pallu

2007-07-01

C-PC (C-phycocyanin) is a water-soluble biliprotein from the filamentous cyanobacterium Spirulina platensis with potent antioxidant, anti-inflammatory and anticancerous properties. In the present study, the effect of C-PC was tested on the proliferation of doxorubicin-sensitive (S-HepG2) and -resistant (R-HepG2) HCC (hepatocellular carcinoma) cell lines. These studies indicate a 50% decrease in the proliferation of S- and R-HepG2 cells treated with 40 and 50 microM C-PC for 24 h respectively. C-PC also enhanced the sensitivity of R-HepG2 cells to doxorubicin. R-HepG2 cells treated with C-PC showed typical apoptotic features such as membrane blebbing and DNA fragmentation. Flow-cytometric analysis of R-HepG2 cells treated with 10, 25 and 50 microM C-PC for 24 h showed 18.8, 39.72 and 65.64% cells in sub-G(0)/G(1)-phase respectively. Cytochrome c release, decrease in membrane potential, caspase 3 activation and PARP [poly(ADP-ribose) polymerase] cleavage were observed in C-PC-treated R-HepG2 cells. These studies also showed down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of the pro-apoptotic Bax (Bcl2-associated X-protein) protein in the R-HepG2 cells treated with C-PC. The present study thus demonstrates that C-PC induces apoptosis in R-HepG2 cells and its potential as an anti-HCC agent.

Chemical characterization of Pleurotus eryngii polysaccharide and its tumor-inhibitory effects against human hepatoblastoma HepG-2 cells.

PubMed

Ren, Daoyuan; Wang, Ning; Guo, Jianjun; Yuan, Li; Yang, Xingbin

2016-03-15

This study was designed to investigate the chemical characterization and antitumor effects of Pleurotus eryngii polysaccharides (PEP). The crude PEP was fractionated into two fractions, namely PEP-1 and PEP-2. HPLC analysis showed that PEP-1 and PEP-2 were heteropolysaccharides mainly composed of glucose with the average molecular weights of 2.54×10(4)Da (PEP-1) and 4.63×10(5)Da (PEP-2), respectively. High molecular mass PEP-2 was shown to exhibit stronger growth inhibition against human hepatoblastoma HepG-2 cells in comparison with PEP-1. Flow cytometric analysis showed that PEP-2 exerted a stimulatory effect on apoptosis of HepG-2 cells, and induced the cell-cycle arrest at the S-phase, with the observation of intracellular ROS production. These findings suggest that the polysaccharides, especially PEP-2, are very important nutritional ingredients responsible for the anticancer health benefits of P. eryngii. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preparative electrophoresis of cultured human cells: Effect of cell cycle phase

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Todd, P. W.; Goolsby, C. L.; Walker, J. T.

1985-01-01

Human epithelioid T-1E cells were cultured in suspension and subjected to density gradient electrophoresis upward in a vertical column. It is indicated that the most rapidly migrating cells were at the beginning of the cell cycle and the most slowly migrating cells were at the end of the cell cycle. The fastest migrating cells divided 24 hr later than the slowest migrating cells. Colonies developing from slowly migrating cells had twice as many cells during exponential growth as did the most rapidly migrating cells, and the numbers of cells per colony at any time was inversely related to the electrophoretic migration rate. The DNA measurements by fluorescence flow cytometry indicates that the slowest migrating cell populations are enriched in cells that have twice as much DNA as the fastest migrating cells. It is concluded that electrophoretic mobility of these cultured human cells declines steadily through the cell cycle and that the mobility is lowest at the end of G sub 2 phase and highest at the beginning of G sub 1 phase.

Mitochondrial Dysfunction and Ca(2+) Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2.

PubMed

Yumnam, Silvia; Hong, Gyeong Eun; Raha, Suchismita; Saralamma, Venu Venkatarame Gowda; Lee, Ho Jeong; Lee, Won-Sup; Kim, Eun-Hee; Kim, Gon Sup

2016-06-01

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells. © 2015 Wiley Periodicals, Inc.

Aberrant activation of ubiquitin D at G2 phase and apoptosis by carcinogens that evoke cell proliferation after 28-day administration in rats.

PubMed

Taniai, Eriko; Yafune, Atsunori; Hayashi, Hitomi; Itahashi, Megu; Hara-Kudo, Yukiko; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

2012-01-01

We have previously reported that renal carcinogens examined in rats increase tubular cell proliferation and topoisomerase (Topo) IIα(+) cells. The present study was aimed at identifying early prediction markers of carcinogens after 28-day treatment in rats. Following gene expression screening by microarrays in renal tubules with renal carcinogens, immunohistochemical analysis and TUNEL-assay were performed with carcinogens targeting different organs. All renal carcinogens tested (ferric nitrilotriacetic acid, ochratoxin A (OTA), monuron, tris(2-chloroethyl) phosphate, and potassium bromate) increased tubular cells immunoreactive for minichromosome maintenance 3 (Mcm3) or ubiquitin D (Ubd) or those showing apoptosis, compared with untreated controls or non-carcinogenic renal toxicants. Carcinogens targeting the liver (thioacetamide (TAA), fenbendazole, piperonyl butoxide (PBO) and methyleugenol), thyroid (sulfadimethoxine), urinary bladder (phenylethyl isothiocyanate), forestomach (butylated hydroxyanisole), glandular stomach (catechol), and colon (chenodeoxycholic acid and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) were examined for induction of Mcm3, Ubd, Topo IIα, Ki-67 and apoptosis using non-carcinogenic toxicants as negative controls. All carcinogens increased Mcm3(+), Ubd(+), Topo IIα(+), Ki-67(+) or TUNEL(+) cells, except for hepatocarcinogen PBO and both colon carcinogens, which did not increase cell proliferation. Ubd(+) cells co-expressing Topo IIα was increased without changing phospho-Histone H3-co-expressing cell population as examined with OTA and TAA. Results revealed cooperative responses of Topo IIα, Ubd and apoptosis by carcinogens inducing high proliferation activity, irrespective of target organs, examined here after a 28-day administration. Aberrant expression of Ubd at G(2) phase and increased apoptosis reflecting aberrant cell cycle regulation may be the common feature of these carcinogens.

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

PubMed

Caro, Andres A; Evans, Kerry L; Cederbaum, Arthur I

2009-01-31

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid, Induces Cytotoxicity, Oxidative Stress, and Apoptosis in HepG2 Cells.

PubMed

Liu, Ying; Cheng, Yajun; Li, Jinwei; Wang, Yuanpeng; Liu, Yuanfa

2018-05-23

In the present study, effects of cis-9,10-epoxy stearic acid (ESA) generated by the thermal oxidation of oleic acid on HepG2 cells, including cytotoxicity, apoptosis, and oxidative stress, were investigated. Our results revealed that ESA decreased the cell viability and induced cell death. Cell cycle analysis with propidium iodide staining showed that ESA induced cell cycle arrest at the G0/G1 phase in HepG2 cells. Cell apoptosis analysis with annexin V and propidium iodide staining demonstrated that ESA induced HepG2 cell apoptotic events in a dose- and time-dependent manner; the apoptosis of cells after treated with 500 μM ESA for 12, 24, and 48 h was 32.16, 38.70, and 65.80%, respectively. Furthermore, ESA treatment to HepG2 cells resulted in an increase in reactive oxygen species and malondialdehyde (from 0.84 ± 0.02 to 8.90 ± 0.50 nmol/mg of protein) levels and a reduction in antioxidant enzyme activity, including superoxide dismutase (from 1.34 ± 0.27 to 0.10 ± 0.007 units/mg of protein), catalase (from 100.04 ± 5.05 to 20.09 ± 3.00 units/mg of protein), and glutathione peroxidase (from 120.44 ± 7.62 to 35.84 ± 5.99 milliunits/mg of protein). These findings provide critical information on the effects of ESA on HepG2 cells, particularly cytotoxicity and oxidative stress, which is important for the evaluation of the biosafety of the oxidative product of oleic acid.

Synergetic effect of functional cadmium–tellurium quantum dots conjugated with gambogic acid for HepG2 cell-labeling and proliferation inhibition

PubMed Central

Xu, Peipei; Li, Jingyuan; Shi, Lixin; Selke, Matthias; Chen, Baoan; Wang, Xuemei

2013-01-01

We prepared and studied novel fluorescent nanocomposites based on gambogic acid (GA) and cadmium–tellurium (CdTe) quantum dots (CdTe QDs) modified with cysteamine for purpose of cancer cell labeling and combined treatment. The nanocomposites were denoted as GA-CdTe. Characterization results indicated that the CdTe QDs can readily bind onto cell plasma membranes and then be internalized into cancer cells for real-time labeling and tracing of human liver hepatocellular carcinoma cell line (HepG2) cells. GA-CdTe significantly enhanced drug accumulation in HepG2 cells and inhibited cancer cell proliferation. GA-CdTe nanocomposites also improved the drug action of GA molecules in HepG2 cells and induced the G2/M phase arrest of the cancer cell cycle, promoting cell apoptosis. Given the sensitive, pH-triggered release of GA-CdTe, the side effects of GA anticancer agents on normal cells/tissues in the blood circulation markedly decreased. Efficient drug release and accumulation in target tumor cells were also facilitated. Thus, the fluorescent GA-CdTe offered a new strategy for potential multimode cancer therapy and provided new channels for research into naturally-active compounds extracted from traditional Chinese medicinal plants. PMID:24109183

Synergetic effect of functional cadmium-tellurium quantum dots conjugated with gambogic acid for HepG2 cell-labeling and proliferation inhibition.

PubMed

Xu, Peipei; Li, Jingyuan; Shi, Lixin; Selke, Matthias; Chen, Baoan; Wang, Xuemei

2013-01-01

We prepared and studied novel fluorescent nanocomposites based on gambogic acid (GA) and cadmium-tellurium (CdTe) quantum dots (CdTe QDs) modified with cysteamine for purpose of cancer cell labeling and combined treatment. The nanocomposites were denoted as GA-CdTe. Characterization results indicated that the CdTe QDs can readily bind onto cell plasma membranes and then be internalized into cancer cells for real-time labeling and tracing of human liver hepatocellular carcinoma cell line (HepG2) cells. GA-CdTe significantly enhanced drug accumulation in HepG2 cells and inhibited cancer cell proliferation. GA-CdTe nanocomposites also improved the drug action of GA molecules in HepG2 cells and induced the G2/M phase arrest of the cancer cell cycle, promoting cell apoptosis. Given the sensitive, pH-triggered release of GA-CdTe, the side effects of GA anticancer agents on normal cells/tissues in the blood circulation markedly decreased. Efficient drug release and accumulation in target tumor cells were also facilitated. Thus, the fluorescent GA-CdTe offered a new strategy for potential multimode cancer therapy and provided new channels for research into naturally-active compounds extracted from traditional Chinese medicinal plants.

Cell partition in two phase polymer systems

NASA Technical Reports Server (NTRS)

Brooks, D. E.

1979-01-01

Aqueous phase-separated polymer solutions can be used as support media for the partition of biological macromolecules, organelles and cells. Cell separations using the technique have proven to be extremely sensitive to cell surface properties but application of the systems are limited to cells or aggregates which do not significantly while the phases are settling. Partition in zero g in principle removes this limitation but an external driving force must be applied to induce the phases to separate since their density difference disappears. We have recently shown that an applied electric field can supply the necessary driving force. We are proposing to utilize the NASA FES to study field-driven phase separation and cell partition on the ground and in zero g to help define the separation/partition process, with the ultimate goal being to develop partition as a zero g cell separation technique.

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

PubMed Central

2013-01-01

Background Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. Results In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. Conclusions The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells PMID:23506616

Angular-dependent light scattering from cancer cells in different phases of the cell cycle.

PubMed

Lin, Xiaogang; Wan, Nan; Weng, Lingdong; Zhou, Yong

2017-10-10

Cancer cells in different phases of the cell cycle result in significant differences in light scattering properties. In order to harvest cancer cells in particular phases of the cell cycle, we cultured cancer cells through the process of synchronization. Flow cytometric analysis was applied to check the results of cell synchronization and prepare for light scattering measurements. Angular-dependent light scattering measurements of cancer cells arrested in the G1, S, and G2 phases have been performed. Based on integral calculations for scattering intensities from 5° to 10° and from 110° to 150°, conclusions have been reached. Clearly, the sizes of the cancer cells in different phases of the cell cycle dominated the forward scatter. Accompanying the increase of cell size with the progression of the cell cycle, the forward scattering intensity also increased. Meanwhile, the DNA content of cancer cells in every phase of the cell cycle is responsible for light scattering at large scatter angles. The higher the DNA content of cancer cells was, the greater the positive effect on the high-scattering intensity. As expected, understanding the relationships between the light scattering from cancer cells and cell cycles will aid in the development of cancer diagnoses. Also, it may assist in the guidance of antineoplastic drugs clinically.

Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

PubMed

Kaldis, Philipp; Aleem, Eiman

2005-11-01

It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings.

A comparison of G2 phase radiation-induced chromatid break kinetics using calyculin-PCC with those obtained using colcemid block.

PubMed

Bryant, Peter E; Mozdarani, Hossein

2007-09-01

To study the possible influence of cell-cycle delay on cells reaching mitosis during conventional radiation-induced chromatid break experiments using colcemid as a blocking agent, we have compared the chromatid break kinetics following a single dose of gamma rays (0.75 Gy) in metaphase CHO cells using calyculin-induced premature chromosome condensation (PCC), with those using colcemid block. Calyculin-induced PCC causes very rapid condensation of G2 cell chromosomes without the need for a cell to progress to mitosis, hence eliminating any effect of cell-cycle checkpoint on chromatid break frequency. We found that the kinetics of the exponential first-order decrease in chromatid breaks with time after irradiation was similar (not significantly different) between the two methods of chromosome condensation. However, use of the calyculin-PCC technique resulted in a slightly increased rate of disappearance of chromatid breaks and thus higher frequencies of breaks at 1.5 and 2.5 h following irradiation. We also report on the effect of the nucleoside analogue ara A on chromatid break kinetics using the two chromosome condensation techniques. Ara A treatment of cells abrogated the decrease in chromatid breaks with time, both using the calyculin-PCC and colcemid methods. We conclude that cell-cycle delay may be a factor determining the absolute frequency of chromatid breaks at various times following irradiation of cells in G2 phase but that the first-order disappearance of chromatid breaks with time and its abrogation by ara A are not significantly influenced by the G2 checkpoint.

[3D evaluation model for drug hepatotoxicity testing on HepG2 cells and its application in drug safety evaluation].

PubMed

Li, Dan-Dan; Tang, Xiang-Lin; Tan, Hong-Ling; Liang, Qian-de; Wang, Yu-Guang; Ma, Zeng-Chun; Xiao, Cheng-Rong; Gao, Yue

2016-04-01

3D in vitro toxicity testing model was developed by magnetic levitation method for culture of the human hepatoma cell line HepG2 and applied to evaluate the drug hepatotoxicity. After formation of stable 3D structure for HepG2 cells, their glycogen storage capacity under 2D and 3D culture conditions were detected by immunohistochemistry technology, and the mRNA expression levels of phase Ⅰ and Ⅱ drug metabolism enzymes, drug transporters, nuclear receptors and liver-specific marker albumin(ALB) were compared between 2D and 3D culture conditions by using RT-PCR method. Immunohistochemistry results showed that HepG2 cells had abundant glycogen storage capacity under 3D culture conditions, which was similar to human liver tissues. The mRNA expression levels of major drug metabolism enzymes, drug transporters, nuclear receptors and ALB in HepG2 cells under 3D culture conditions were up-regulated as compared with 2D culture conditions. For drug hepatotoxicity evaluation, the typical hepatotoxic drug acetaminophen(APAP), and most reported drugs Polygonum multiflorum Thunb.(Chinese name He-shou-wu) and Psoraleae corylifolia L.(Chinese name Bu-gu-zhi) were selected for single dose and repeated dose(7 d) exposure. In the repeated dose exposure test, 3D HepG2 cells showed higher sensitivity. This established 3D HepG2 cells model with magnetic levitation 3D culture techniques was more close to the human liver tissues both in morphology and functions, so it was a better 3D hepatotoxicity evaluation model. Copyright© by the Chinese Pharmaceutical Association.

HepG2 human hepatocarcinomas cells sensitization by endogenous porphyrins

NASA Astrophysics Data System (ADS)

Vonarx-Coinsmann, Veronique; Foultier, Marie-Therese; de Brito, Leonor X.; Morlet, Laurent; Patrice, Thierry

1995-03-01

We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

Exploring a Link Between NF-KB and G2/M Cell Cycle Arrest in Breast Cancer Cells

DTIC Science & Technology

2005-04-01

studies with esophageal squamous cell carcinom a lines have shown that IR induced p21waf1/ ciP ’ and a G2 cell cycle arrest that could als o be...i AD Award Number : DAMD17-02-1-062 3 TITLE : Exploring a Link Between NF-KB and G 2 /M Cell Cycle Arres t in Breast Cancer Cell s PRINCIPAL...Mar 2005 ) 4 . TITLE AND SUBTITL E Exploring a Link Between NF-kB and G 2 /M Cell Cycle Arres t in Breast Cancer Cells 5. FUND/NG NUMBERS DAMD17-02-1

Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.

PubMed

Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Mason, Ronald P

2015-01-01

Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA. Copyright © 2014 Elsevier Inc. All rights reserved.

Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S-phase transition.

PubMed

Ghule, Prachi N; Seward, David J; Fritz, Andrew J; Boyd, Joseph R; van Wijnen, Andre J; Lian, Jane B; Stein, Janet L; Stein, Gary S

2018-05-10

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3-dimensional context of nuclear organization is reflected by two well documented observations. DNA replication-dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone-related requirements for G1/S-phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci. © 2018 Wiley Periodicals, Inc.

The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

PubMed Central

Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

2013-01-01

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

Structure related effects of flavonoid aglycones on cell cycle progression of HepG2 cells: Metabolic activation of fisetin and quercetin by catechol-O-methyltransferase (COMT).

PubMed

Poór, Miklós; Zrínyi, Zita; Kőszegi, Tamás

2016-10-01

Dietary flavonoids are abundant in the Plant Kingdom and they are extensively studied because of their manifold pharmacological activities. Recent studies highlighted that cell cycle arrest plays a key role in their antiproliferative effect in different tumor cells. However, structure-activity relationship of flavonoids is poorly characterized. In our study the influence of 18 flavonoid aglycones (as well as two metabolites) on cell cycle distribution was investigated. Since flavonoids are extensively metabolized by liver cells, HepG2 tumor cell line was applied, considering the potential metabolic activation/inactivation of flavonoids. Our major observations are the followings: (1) Among the tested compounds diosmetin, fisetin, apigenin, lutelin, and quercetin provoked spectacular extent of G2/M phase cell cycle arrest. (2) Inhibition of catechol-O-methyltransferase enzyme by entacapone decreased the antiproliferative effects of fisetin and quercetin. (3) Geraldol and isorhamnetin (3'-O-methylated metabolites of fisetin and quercetin, respectively) demonstrated significantly higher antiproliferative effect on HepG2 cells compared to the parent compounds. Based on these results, O-methylated flavonoid metabolites or their chemically modified derivatives may be suitable candidates of tumor therapy in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

[Overexpression of SEPP1 inhibits the proliferation and induces cell cycle G2/M arrest of 786-O and 769-P human renal carcinoma cells].

PubMed

Liu, Kan; Zhao, Chaofei; Chen, Jianwen; Wu, Shengpan; Yao, Yuanxin; Wu, Chong; Luo, Guoxiong; Zhang, Xu

2016-06-01

Objective To establish selenoprotein P, plasma 1 (SEPP1) gene recombinant lentiviral vector and investigate the effect of SEPP1 on the proliferation of human clear cell renal cell carcinoma (ccRCC) cells. Methods cDNA sequence of SEPP1 was cloned from the total cDNA of HEK293T cells by PCR. Then, the cDNA fragment was combined with the pLV-EGFP(2A)Puro vector and the constructed plasmid pLV-EGFP(2A)Puro-SEPP1 was transfected into HEK293T cells for packaging the virus. Forty-eight hours after transfected with the virus supernatant, the level of SEPP1 protein in 769-P and 786-O cells were tested by Western blotting. Cells were divided into recombinant lentivirus-infected cells, empty vector lentivirus-infected cells and the blank control cells. Cell proliferation rate was detected by MTS assay, colony forming ability was evaluated by plate clony formation assay and cell cycle change was assayed by flow cytometry after transfected with pLV-EGFP(2A)Puro-SEPP1 or empty pLV-EGFP(2A)Puro vector. Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 was constructed successfully. After being infected by the virus supernatant, the 786-O and 769-P cells expressed EGFP. Compared with the empty vector group and the blank control group, expression level of SEPP1 in the experimental group was much higher. The cell proliferative ability was inhibited in the cells overexpressing SEPP1, and the colony forming ability of SEPP1-overexpressed cells evidently decreased. Cell cycle was arrested in G2/M phase in 786-O cells overexpressing SEPP1. Conclusion The recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 has been constructed successfully. Overexpression of SEPP1 could significantly reduce the proliferation rate of 786-O and 769P cells, and cause G2/M phase arrest of 786-O cells.

VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Zhitao; Lu, Xiao; Zhu, Ping

Highlights: Black-Right-Pointing-Pointer VCC-1 is hypothesized to be associated with carcinogenesis. Black-Right-Pointing-Pointer Levels of VCC-1 are increased significantly in HCC. Black-Right-Pointing-Pointer Over-expression of VCC-1 could promotes cellular proliferation rate. Black-Right-Pointing-Pointer Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. Black-Right-Pointing-Pointer VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellularmore » carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.« less

The hormesis effect of BDE-47 in HepG2 cells and the potential molecular mechanism.

PubMed

Wang, Liulin; Zou, Wen; Zhong, Yufang; An, Jing; Zhang, Xinyu; Wu, Minghong; Yu, Zhiqiang

2012-03-07

Polybrominated diphenyl ethers (PBDEs) had been used extensively in electrical and electronic products as brominated flame retardants. PBDEs are widely distributed in environment media and wildlife since they are lipophilic and persistent, resulting in bioaccumulation and bioamplification through food chains. Accumulation of PBDEs in the environment and human tissues will consequently cause potential negative effects on the ecological environment and human health. To date, some in vitro and in vivo studies have reported that PBDEs possess neurotoxicity, hepatotoxicity, immunotoxicity, reproduction toxicity, endocrine disrupting activity and carcinogenicity. BDE-47 is one of the most predominant PBDE congeners detected in human tissues. The objective of this study is to investigate whether low concentration of BDE-47 could cause hormesis effect in the human hepatoma HepG(2) cells, and to explore the possible molecular mechanism. The results showed that low concentration of BDE-47 (10(-10), 10(-9) and 10(-8) M) could promote cell proliferation and cause no obvious change in DNA damage or cell apoptosis, while the high concentration significantly inhibit cell proliferation. Meanwhile, the reactive oxygen species (ROS) in low concentration BDE-47 (10(-10), 10(-9) and 10(-8) M) treated groups significantly elevated compared with the control group. After low concentration BDE-47 treatment, the expression of proliferating cell nuclear antigen (PCNA), Cyclin D1, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylated protein kinase B (p-Akt) in the HepG(2) cells was markedly up-regulated. However, in DNA-PKcs inhibited cells, the promotion effect on cell proliferation was significantly suppressed. Cell cycle analysis showed a significant decrease in G1 phase after exposure to low concentration of BDE-47. Moreover, pre-exposure to low concentration BDE-47 seemed alleviate the negative effects of high concentration (50 μM) exposure to cause DNA damage

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

PubMed Central

Barr, Alexis R.; Cooper, Samuel; Heldt, Frank S.; Butera, Francesca; Stoy, Henriette; Mansfeld, Jörg; Novák, Béla; Bakal, Chris

2017-01-01

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability. PMID:28317845

Zeatin is indispensable for the G2-M transition in tobacco BY-2 cells.

PubMed

Laureys, F; Dewitte, W; Witters, E; Van Montagu, M; Inzé, D; Van Onckelen, H

1998-04-10

The importance of N6-isoprenoid cytokinins in the G2-M transition of Nicotiana tabacum BY-2 cells was investigated. Both cytokinin biosynthesis and entry in mitosis were partially blocked by application at early or late G2 of lovastatin (10 microM), an inhibitor of mevalonic acid synthesis. LC-MS/MS quantification of endogenous cytokinins proved that lovastatin affects cytokinin biosynthesis by inhibiting HMG-CoA reductase. Out of eight different aminopurines and a synthetic auxin tested for their ability to override lovastatin inhibition of mitosis, only zeatin was active. Our data point to a key role for a well-defined cytokinin (here, zeatin) in the G2-M transition of tobacco BY-2 cells.

Comparative analysis of 3D culture methods on human HepG2 cells.

PubMed

Luckert, Claudia; Schulz, Christina; Lehmann, Nadja; Thomas, Maria; Hofmann, Ute; Hammad, Seddik; Hengstler, Jan G; Braeuning, Albert; Lampen, Alfonso; Hessel, Stefanie

2017-01-01

Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

Origin and fate of lymphocytic choriomeningitis virus-specific CD8+ T cells coexpressing the inhibitory NK cell receptor Ly49G2.

PubMed

Peacock, Craig D; Welsh, Raymond M

2004-07-01

CD8+ T cells that coexpress the inhibitory NK cell receptor, Ly49G2 (G2), are present in immunologically naive C57BL/6 mice but display Ags found on memory T cells. To assess how G2+CD8+ cells relate to bona fide memory cells, we examined the origin and fate of lymphocytic choriomeningitis virus (LCMV)-induced G2+CD8+ cells. During early (day 4) acute LCMV infection, both G2+ and G2-CD8+ T cell subsets underwent an attrition in number and displayed an activation (CD69(high)1B11(high)CD62L(low)) phenotype. By day 8, both subsets synthesized IFN-gamma in response to immunodominant LCMV peptides, though the expansion of G2+ cells was less than that of G2- cells. Adoptive transfer experiments with purified G2- or G2+CD8+ cells from naive mice indicated that the LCMV-specific G2+ subset was derived from a pre-existing G2+ population and not generated from G2- cells responding to LCMV infection. Their participation in the LCMV-specific T cell response increased with age, reflecting an increase in the size of the pre-existing G2+ pool. Following establishment of stable LCMV memory, the proportion of CD8+ cells coexpressing G2 was reduced in comparison to naive controls, presumably due to displacement by G2- LCMV-specific memory cells. LCMV-specific G2+ cells were present in the memory pool, but at low frequencies, and they did not exhibit the typical phenotypic changes of reactivation during secondary challenge. We suggest that G2+CD8+ cells represent a cell lineage distinct from bona fide memory T cells, but that they can participate in an acute virus-specific T cell response.

Low doses of GM-CSF (molgramostim) and G-CSF (filgrastim) after cyclophosphamide (4 g/m2) enhance the peripheral blood progenitor cell harvest: results of two randomized studies including 120 patients

PubMed Central

Quittet, Philippe; Ceballos, Patrice; Lopez, Ernesto; Lu, Zhao-Yang; Latry, Pascal; Becht, Catherine; Legouffe, Eric; Fegueux, Nathalie; Exbrayat, Carole; Pouessel, Damien; Rouillé, Valérie; Daures, Jean-Pierre; Klein, Bernard; Rossi, Jean-François

2006-01-01

The use of a combination of G-CSF and GM-CSF to G-CSF alone, after cyclophosphamide (4g/m2) was compared in 2 randomized phase III studies, including 120 patients. In study A, 60 patients received 5 × 2 μg/kg/day of G-CSF and GM-CSF compared to 5 μg/kg/day of G-CSF. In study B, 60 patients received 2.5 × 2 μg/kg/day G-CSF and GM-CSF compared to G-CSF alone (5 μg/kg/day). With the aim to collect at least 5 × 106/kg CD34 cells in a maximum of 3 large volume leukapherisis (LK), 123 LK were performed in study A, showing significant higher number of patients reaching 10 × 106/kg CD34 cells (21/29 in G+GM-CSF arm vs 11/27 in G-CSF arm, P= .00006). In study B, 109 LK were performed, with similar results (10/27 vs 15/26, P= .003). In both the study, the total harvest of CD34 cells/kg was 2-fold higher in G-CSF plus GM-CSF group (18.3 × 106 in study A and 15.85 × 106 in study B) than in G-CSF group (9 × 106 in study A and 8.1 × 106 in study B), a difference particularly seen in multiple myeloma, with no significant difference in terms of mobilized myeloma cells between G-CSF and GM-CSF groups. PMID:16883311

Cancer cells mimic in vivo spatial-temporal cell-cycle phase distribution and chemosensitivity in 3-dimensional Gelfoam® histoculture but not 2-dimensional culture as visualized with real-time FUCCI imaging.

PubMed

Yano, Shuya; Miwa, Shinji; Mii, Sumiyuki; Hiroshima, Yukihiko; Uehara, Fuminaru; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Zhao, Ming; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M

2015-01-01

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We previously reported monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor, intravitally in live mice, using a fluorescence ubiquitination-based cell-cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after cessation of chemotherapy. These results suggested why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. In the present report, we used FUCCI imaging and Gelfoam® collagen-sponge-gel histoculture, to demonstrate in real time, that the cell-cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is highly similar, whereby only the surface cells proliferate and interior cells are quiescent in G0/G1. This is in contrast to 2D culture where most cancer cells cycle. Similarly, the cancer cells responded similarly to toxic chemotherapy in Gelfoam® culture as in vivo, and very differently than cancer cells in 2D culture which were much more chemosensitive. Gelfoam® culture of FUCCI-expressing cancer cells offers the opportunity to image the cell cycle of cancer cells continuously and to screen for novel effective therapies to target quiescent cells, which are the majority in a tumor and which would have a strong probability to be effective in vivo.

Ammonium Ion Binding to DNA G-Quadruplexes: Do Electrospray Mass Spectra Faithfully Reflect the Solution-Phase Species?

NASA Astrophysics Data System (ADS)

Balthasart, Françoise; Plavec, Janez; Gabelica, Valérie

2013-01-01

G-quadruplex nucleic acids can bind ammonium ions in solution, and these complexes can be detected by electrospray mass spectrometry (ESI-MS). However, because ammonium ions are volatile, the extent to which ESI-MS quantitatively could provide an accurate reflection of such solution-phase equilibria is unclear. Here we studied five G-quadruplexes having known solution-phase structure and ammonium ion binding constants: the bimolecular G-quadruplexes (dG4T4G4)2, (dG4T3G4)2, and (dG3T4G4)2, and the intramolecular G-quadruplexes dG4(T4G4)3 and dG2T2G2TGTG2T2G2 (thrombin binding aptamer). We found that not all mass spectrometers are equally suited to reflect the solution phase species. Ion activation can occur in the electrospray source, or in a high-pressure traveling wave ion mobility cell. When the softest instrumental conditions are used, ammonium ions bound between G-quartets, but also additional ammonium ions bound at specific sites outside the external G-quartets, can be observed. However, even specifically bound ammonium ions are in some instances too labile to be fully retained in the gas phase structures, and although the ammonium ion distribution observed by ESI-MS shows biases at specific stoichiometries, the relative abundances in solution are not always faithfully reflected. Ion mobility spectrometry results show that all inter-quartet ammonium ions are necessary to preserve the G-quadruplex fold in the gas phase. Ion mobility experiments, therefore, help assign the number of inner ammonium ions in the solution phase structure.[Figure not available: see fulltext.

Inferring Toxicological Responses of HepG2 Cells from ...

EPA Pesticide Factsheets

Understanding the dynamic perturbation of cell states by chemicals can aid in for predicting their adverse effects. High-content imaging (HCI) was used to measure the state of HepG2 cells over three time points (1, 24, and 72 h) in response to 976 ToxCast chemicals for 10 different concentrations (0.39-200µM). Cell state was characterized by p53 activation (p53), c-Jun activation (SK), phospho-Histone H2A.x (OS), phospho-Histone H3 (MA), alpha tubulin (Mt), mitochondrial membrane potential (MMP), mitochondrial mass (MM), cell cycle arrest (CCA), nuclear size (NS) and cell number (CN). Dynamic cell state perturbations due to each chemical concentration were utilized to infer coarse-grained dependencies between cellular functions as Boolean networks (BNs). BNs were inferred from data in two steps. First, the data for each state variable were discretized into changed/active (> 1 standard deviation), and unchanged/inactive values. Second, the discretized data were used to learn Boolean relationships between variables. In our case, a BN is a wiring diagram between nodes that represent 10 previously described observable phenotypes. Functional relationships between nodes were represented as Boolean functions. We found that inferred BN show that HepG2 cell response is chemical and concentration specific. We observed presence of both point and cycle BN attractors. In addition, there are instances where Boolean functions were not found. We believe that this may be either

Akt interaction with PLC(gamma) regulates the G(2)/M transition triggered by FGF receptors from MDA-MB-231 breast cancer cells.

PubMed

Browaeys-Poly, Edith; Perdereau, Dominique; Lescuyer, Arlette; Burnol, Anne-Françoise; Cailliau, Katia

2009-12-01

Estrogen-independent breast cancer cell growth is under the control of fibroblast growth factors receptors (FGFRs), but the role of phospholipase C gamma (PLC(gamma)) and Akt, the downstream effectors activated by FGFRs, in cell proliferation is still unresolved. FGFRs from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a powerful model system to assess the G(2)/M checkpoint regulation. Under FGF1 stimulation, an analysis of the progression in the M-phase of the cell cycle and of the Akt signaling cascades were performed using the phosphatidylinositol-3-kinase inhibitor, LY294002, and a mimetic peptide of the SH3 domain of PLC(gamma). Activated Akt binds and phosphorylates PLC(gamma) before Akt targets the tumor suppressor Chfr. Disruption of the Akt-PLC(gamma) interaction directs Akt binding to Chfr and accelerates the alleviation of the G(2)/M checkpoint. The PLC(gamma)-Akt interaction, triggered by FGF receptors from estrogen-independent breast cancer cells MDA-MB-231, regulates progression in the M-phase of the cell cycle.

Fungal 7-epi-10-deacetyltaxol produced by an endophytic Pestalotiopsis microspora induces apoptosis in human hepatocellular carcinoma cell line (HepG2).

PubMed

Subban, Kamalraj; Singh, Satpal; Subramani, Ramesh; Johnpaul, Muthumary; Chelliah, Jayabaskaran

2017-11-28

Paclitaxel (taxol) is a potent anticancer drug that is used in the treatment of a wide variety of cancerous. In the present study, we identified a taxol derivative named 7-epi-10-deacetyltaxol (EDT) from the culture of an endophytic fungus Pestalotiopsis microspora isolated from the bark of Taxodium mucronatum. This study was carried out to investigate the effects of fungal EDT on cell proliferation, the induction of apoptosis and the molecular mechanisms of apoptosis in human hepatoma HepG2 cells in vitro. The endophytic fungus was identified by traditional and molecular taxonomical characterization and the fungal EDT was purified using column chromatography and confirmed by various spectroscopic and chromatographic comparisons with authentic paclitaxel. We studied the in vitro effects of EDT on HepG2 cells for parameters such as cell cycle distribution, DNA fragmentation, reactive oxygen species (ROS) generation and nuclear morphology. Further, western blot analysis was used to evaluate Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), p38-mitogen activated protein kinase (MAPK) and poly [ADP-ribose] polymerase (PARP) expression. We demonstrate that the fungal EDT exhibited significant in vitro cytotoxicity in HepG2 cells. We investigated cytotoxicity mechanism of EDT in HepG2 cells. The results showed nuclear condensation and DNA fragmentation were observed in cells treated with fungal EDT. Besides, the fungal EDT arrested HepG2 cells at G2/M phase of cell cycle. Furthermore, fungal EDT induced apoptosis in HepG2 cells in a dose-dependent manner associated with ROS generation and increased Bax/Bcl-2 ratio, p38 MAPKs and PARP cleavage. Our data show that EDT induced apoptotic cell death in HepG2 cells occurs through intrinsic pathway by generation of ROS mediated and activation of MAPK pathway. This is the first report for 7-epi-10-deacetyltaxol (EDT) isolated from a microbial source.

Action of caffeine on x-irradiated HeLa cells. III. enhancement of x-ray-induced killing during G/sub 2/ arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busse, P.M.; Bose, S.K.; Jones, R.W.

1978-11-01

The ability of caffeine to enhance the expression of potentially lethal x-ray damage in HeLa S3 cells was examined as a function of the age of the cells in the generation cycle. Synchronous populations were irradiated at different times after mitotic collection and treated for various intervals with 1 mM caffeiene, which causes negligible killing of unirradiated cells. The response was thereby determined as a function of cell age at both the time of irradiation and the time of exposure to caffeine. The amount of cell killing depends strongly on when in the cycle caffeine is present and only weaklymore » on when the cells are irradiated. If cells are irradiated in early G/sub 1/, caffeine treatment enhances killing for 2 to 3 hr. No additional enhancement is observed until 16 to 17 hr postcollection, corresponding to G/sub 2/; here they enter a second period of much greater sensitivity. Similarly, fluorodeoxyuridine resynchronized cells irradiated during S and treated with caffeine suffer no enhanced killing until they pass into this sensitive phase in G/sub 2/, approximately 7 hr after release from the fluorodeoxyuridine block. The sensitive period appears to coincide with G/sub 2/ arrest. The rate and extent of killing during this period are dependent upon the x-ray dose and the caffeine concentration. In the absence of caffeine, cells irradiated in G/sub 1/ lose sensitivity to caffeine in about 9 hr; they do so faster in G/sub 2/. It is concluded that the potentially lethal x-ray damage expressed on treatment with caffeine is retained for many hours in the presence of caffeine and is maximally manifested by G/sub 2/-arrested cells.« less

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

PubMed Central

Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

2008-01-01

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

PubMed Central

Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

2012-01-01

Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study. PMID:22655088

Chip-based magnetic solid phase microextraction coupled with ICP-MS for the determination of Cd and Se in HepG2 cells incubated with CdSe quantum dots.

PubMed

Yu, Xiaoxiao; Chen, Beibei; He, Man; Wang, Han; Hu, Bin

2018-03-01

The quantification of trace Cd and Se in cells incubated with CdSe quantum dots (QDs) is critical to investigate the cytotoxicity of CdSe QDs. In this work, a miniaturized platform, namely chip-based magnetic solid phase microextraction (MSPME) packing with sulfhydryl group functionalized magnetic nanoparticles, was fabricated and combined with inductively coupled plasma mass spectrometry (ICP-MS) for the determination of trace Cd and Se in cells. Under the optimized conditions, the limits of detection (LOD) of the developed chip-based MSPME-ICP-MS system are 2.2 and 21ngL -1 for Cd and Se, respectively. The proposed method is applied successfully to the analysis of total and released small molecular fraction of Cd and Se in Human hepatocellular carcinoma cells (HepG2 cells) incubated with CdSe QDs, and the recoveries for the spiked samples are in the range of 86.0-109%. This method shows great promise to analyze cell samples and the obtained results are instructive to explore the cytotoxicity mechanism of CdSe QDs in cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Ethyl acetate fraction of Garcina epunctata induces apoptosis in human promyelocytic cells (HL-60) through the ROS generation and G0/G1 cell cycle arrest: a bioassay-guided approach.

PubMed

Constant Anatole, Pieme; Guru, Santoh Kumar; Bathelemy, Ngamegni; Jeanne, Ngogang; Bhushan, Shashi; Murayama, Tetsuya; Saxena, Ajit Kumar

2013-11-01

Number of deaths due to cancer diseases is increasing in the world. There is an urgent need to develop alternative therapeutic measures against the disease. Our study reports the cytotoxicity activity of Garcina epunctata (gutifferae) in human promyelocytic leukemia cells (HL-60) and prostate cancer cells (PC-3) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and morphological changes associated with apoptosis were examined by flow cytometry and Hoescht staining respectively. The results of in vitro antiproliferative screening of fractions and extract from G. epunctata indicated that three fractions inhibited the viability of PC-3 cells with IC₅₀ varied from 50 to 88 μ/ml while two fractions inhibited the proliferation of HL-60 cells with IC₅₀ range between 47.5 and 12 μg/ml. Among the entire fraction tested, Hex-EtOAc (75:25) showed cytotoxic effects on the two cell lines and EtOAc fraction was most active only HL-60 cells (12 μg/ml). Treatment of HL-60 cells with G. epunctata (20, 50, 100 μg/ml) for 24 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a number of apoptotic bodies containing nuclear fragments were observed in cells treated with 100 μg/ml. The EtOAc fraction of G. epunctata treatment significantly arrested HL-60 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated as well as the loss of membrane mitochondrial potential in a concentration dependant manner. The results demonstrated that the EtOAc fraction of G. epunctata inhibited the proliferation of HL-60 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

PubMed Central

2010-01-01

Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be

Cytostatic and genotoxic effect of temephos in human lymphocytes and HepG2 cells.

PubMed

Benitez-Trinidad, A B; Herrera-Moreno, J F; Vázquez-Estrada, G; Verdín-Betancourt, F A; Sordo, M; Ostrosky-Wegman, P; Bernal-Hernández, Y Y; Medina-Díaz, I M; Barrón-Vivanco, B S; Robledo-Marenco, M L; Salazar, A M; Rojas-García, A E

2015-06-01

Temephos is an organophosphorus pesticide that is used in control campaigns against Aedes aegypti mosquitoes, which transmit dengue. In spite of the widespread use of temephos, few studies have examined its genotoxic potential. The aim of this study was to evaluate the cytotoxic, cytostatic and genotoxic effects of temephos in human lymphocytes and hepatoma cells (HepG2). The cytotoxicity was evaluated with simultaneous staining (FDA/EtBr). The cytostatic and genotoxic effects were evaluated using comet assays and the micronucleus technique. We found that temephos was not cytotoxic in either lymphocytes or HepG2 cells. Regarding the cytostatic effect in human lymphocytes, temephos (10 μM) caused a significant decrease in the percentage of binucleated cells and in the nuclear division index as well as an increase in the apoptotic cell frequency, which was not the case for HepG2 cells. The comet assay showed that temephos increased the DNA damage levels in human lymphocytes, but it did not increase the MN frequency. In contrast, in HepG2 cells, temephos increased the tail length, tail moment and MN frequency in HepG2 cells compared to control cells. In conclusion, temephos causes stable DNA damage in HepG2 cells but not in human lymphocytes. These findings suggest the importance of temephos biotransformation in its genotoxic effect. Copyright © 2015. Published by Elsevier Ltd.

ANT2 expression under hypoxic conditions produces opposite cell-cycle behavior in 143B and HepG2 cancer cells.

PubMed

Chevrollier, Arnaud; Loiseau, Dominique; Gautier, Fabien; Malthièry, Yves; Stepien, Georges

2005-01-01

Under hypoxic conditions, mitochondrial ATP production ceases, leaving cells entirely dependent on their glycolytic metabolism. The cytoplasmic and intramitochondrial ATP/ADP ratios, partly controlled by the adenine nucleotide translocator (ANT), are drastically modified. In dividing and growing cells that have a predominantly glycolytic metabolism, the ANT isoform 2, which has kinetic properties allowing ATP import into mitochondria, is over-expressed in comparison to control cells. We studied the cellular metabolic and proliferative response to hypoxia in two transformed human cell lines with different metabolic backgrounds: HepG2 and 143B, and in their rho(o) derivatives, i.e., cells with no mitochondrial DNA. Transformed 143B and rho(o) cells continued their proliferation whereas HepG2 cells, with a more differentiated phenotype, arrested their cell-cycle at the G(1)/S checkpoint. Hypoxia induced an increase in glycolytic activity, correlated to an induction of VEGF and hexokinase II (HK II) expression. Thus, according to their tumorigenicity, transformed cells may adopt one of two distinct behaviors to support hypoxic stress, i.e., proliferation or quiescence. Our study links the constitutive glycolytic activity and ANT2 expression levels of transformed cells with the loss of cell-cycle control after oxygen deprivation. ATP import by ANT2 allows cells to maintain their mitochondrial integrity while acquiring insensitivity to any alterations in the proteins involved in oxidative phosphorylation. This loss of cell dependence on oxidative metabolism is an important factor in the development of tumors.

Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms.

PubMed

Yu, Si; Wang, Lijiao; Cao, Zhixing; Gong, Daoyin; Liang, Qianyi; Chen, Hanting; Fu, Huizhu; Wang, Wenwen; Tang, Xue; Xie, Zihao; He, Yang; Peng, Cheng; Li, Yuzhi

2018-06-01

Polyphyllin Ι is a steroidal saponin isolated from the rhizoma of Paris polyphylla. In the present study, we aimed to investigate the anticancer effects of polyphyllin Ι in colorectal cancer and to elucidate the potential underlying molecular mechanisms. Using, CCK8 assay, flow cytometry, laser confocal microscope analysis and western blot, the anticancer effects of the polyphyllin Ι were analysed in colorectal cells. Our results indicate that polyphyllin Ι significantly decreased cell viability of HCT 116 cells and induced autophagy. Furthermore, we found that polyphyllin Ι induced autophagy in an ROS-dependent cell death and not related with PI3 K/AKT/mTOR pathway. We also provide evidence that excessive ROS triggered by polyphyllin Ι could induce G2/M phase arrest via regulating cycle proteins expression of cell cycle regulators, such as p21 and cyclinB1. In conclusion, polyphyllin Ι exhibit anticancer effect through ROS-dependent autophagy and induces G2/M arrest in colorectal cancer.

TLR3 dsRNA agonist inhibits growth and invasion of HepG2.2.15 HCC cells.

PubMed

Chen, Li; Xu, Yu-Yin; Zhou, Jia-Ming; Wu, Yuan-Yuan; E, Qun; Zhu, Yuan-Yuan

2012-07-01

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). TLR3 expression and function in cancer cells are not well understood. In this study, we investigated whether TLR3 agonist dsRNA (BM-06) can inhibit proliferation and invasion, and promote apoptosis in HepG2.2.15 cells. HepG2.2.15 cells secreting hepatitis B virus (HBV) were treated with BM-06 and poly(I:C). Western blot analysis and PCR were employed to determine pharmacodynamic changes in biomarkers relevant to TLR3 signaling. Cell proliferation, invasion and apoptosis were analyzed by CCK-8 assay, transwell assay and flow cytometry. The expression of HBsAg, and HBcAg was observed by immunohistochemistry. Compared with untreated cells, pharmacological NF-κB activity of the TLR3 pathway by BM-06 (1.734-fold) or poly(I:C) (1.377-fold) was induced. By western blot analysis, we found that dsRNA induced TLR3-activated HepG2.2.15 cells which expressed NF-κB levels predominantly in the cytoplasmic fraction but fewer signals in the nucleus. BM-06 inhibited the proliferation, invasion and secretion of HBV, and induced apoptosis in HepG2.2.15 cells. In addition, the antitumor effects of BM-06 were superior to poly(I:C). Pharmacological activation of the TLR3 pathway by BM-06 can inhibit HepG2.2.15 cell growth.

Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horiuchi, Shinji; Kato, Kiyoko; Suga, Shin

2005-05-01

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phasemore » of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.« less

Spliceosomal protein E regulates neoplastic cell growth by modulating expression of cyclin E/CDK2 and G2/M checkpoint proteins.

PubMed

Li, Z; Pützer, B M

2008-12-01

Small nuclear ribonucleoproteins are essential splicing factors. We previously identified the spliceosomal protein E (SmE) as a downstream effector of E2F1 in p53-deficient human carcinoma cells. Here, we investigated the biological relevance of SmE in determining the fate of cancer and non-tumourigenic cells. Adenovirus-mediated expression of SmE selectively reduces growth of cancerous cells due to decreased cell proliferation but not apoptosis. A similar growth inhibitory effect for SmD1 suggests that this is a general function of Sm-family members. Deletion of Sm-motifs reveals the importance of the Sm-1 domain for growth suppression. Consistently, SmE overexpression leads to inhibition of DNA synthesis and G2 arrest as shown by BrdU-incorporation and MPM2-staining. Real-time RT-PCR and immunoblotting showed that growth arrest by SmE directly correlates with the reduction of cyclin E, CDK2, CDC25C and CDC2 expression, and up-regulation of p27Kip. Importantly, SmE activity was not associated with enhanced expression of other spliceosome components such as U1 SnRNP70, suggesting that the growth inhibitory effect of SmE is distinct from its pre-mRNA splicing function. Furthermore, specific inactivation of SmE by shRNA significantly increased the percentage of cells in S phase, whereas the amount of G2/M arrested cells was reduced. Our data provide evidence that Sm proteins function as suppressors of tumour cell growth and may have major implications as cancer therapeutics.

Jaceosidin, isolated from dietary mugwort (Artemisia princeps), induces G2/M cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation.

PubMed

Lee, Jong-Gyu; Kim, Ji-Hyun; Ahn, Ji-Hye; Lee, Kyung-Tae; Baek, Nam-In; Choi, Jung-Hye

2013-05-01

Jaceosidin, a flavonoid derived from Artemisia princeps (Japanese mugwort), has been shown to inhibit the growth of several human cancer cells, However, the exact mechanism for the cytotoxic effect of jaceosidin is not completely understood. In this study, we investigated the molecular mechanism involved in the antiproliferative effect of jaceosidin in human endometrial cancer cells. We demonstrated that jaceosidin is a more potent inhibitor of cell growth than cisplatin in human endometrial cancer cells. In contrast, jaceosidin-induced cytotoxicity in normal endometrial cells was lower than that observed for cisplatin. Jaceosidin induced G2/M phase cell cycle arrest and modulated the levels of cyclin B and p-Cdc2 in Hec1A cells. Knockdown of p21 using specific siRNAs partially abrogated jaceosidin-induced cell growth inhibition. Additional mechanistic studies revealed that jaceosidin treatment resulted in an increase in phosphorylation of Cdc25C and ATM-Chk1/2. Ku55933, an ATM inhibitor, reversed jaceosidin-induced cell growth inhibition, in part. Moreover, jaceosidin treatment resulted in phosphorylation of ERK, and pretreatment with the ERK inhibitor, PD98059, attenuated cell growth inhibition by jaceosidin. These data suggest that jaceosidin, isolated from Japanese mugwort, modulates the ERK/ATM/Chk1/2 pathway, leading to inactivation of the Cdc2-cyclin B1 complex, followed by G2/M cell cycle arrest in endometrial cancer cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

PubMed

Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

2016-01-01

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

PubMed Central

Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

2007-01-01

Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

PubMed

Cao, Liyan; Cheng, Shan; Du, Juan; Guo, Yanhai; Huang, Xiaofeng

2017-04-01

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H 2 O 2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H 2 O 2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H 2 O 2 . Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

FOXC2 regulates the G2/M transition of stem cell-rich breast cancer cells and sensitizes them to PLK1 inhibition

PubMed Central

Pietilä, Mika; Vijay, Geraldine V.; Soundararajan, Rama; Yu, Xian; Symmans, William F.; Sphyris, Nathalie; Mani, Sendurai A.

2016-01-01

Cancer cells with stem cell properties (CSCs) underpin the chemotherapy resistance and high therapeutic failure of triple-negative breast cancers (TNBCs). Even though CSCs are known to proliferate more slowly, they are sensitive to inhibitors of G2/M kinases such as polo-like kinase 1 (PLK1). Understanding the cell cycle regulatory mechanisms of CSCs will help target these cells more efficiently. Herein, we identify a novel role for the transcription factor FOXC2, which is mostly expressed in CSCs, in the regulation of cell cycle of CSC-enriched breast cancer cells. We demonstrate that FOXC2 expression is regulated in a cell cycle-dependent manner, with FOXC2 protein levels accumulating in G2, and rapidly decreasing during mitosis. Knockdown of FOXC2 in CSC-enriched TNBC cells delays mitotic entry without significantly affecting the overall proliferation rate of these cells. Moreover, PLK1 activity is important for FOXC2 protein stability, since PLK1 inhibition reduces FOXC2 protein levels. Indeed, FOXC2 expressing CSC-enriched TNBC cells are sensitive to PLK1 inhibition. Collectively, our findings demonstrate a novel role for FOXC2 as a regulator of the G2/M transition and elucidate the reason for the observed sensitivity of CSC-enriched breast cancer cells to PLK1 inhibitor. PMID:27064522

Surface grafted glycopolymer brushes to enhance selective adhesion of HepG2 cells.

PubMed

Chernyy, Sergey; Jensen, Bettina E B; Shimizu, Kyoko; Ceccato, Marcel; Pedersen, Steen Uttrup; Zelikin, Alexander N; Daasbjerg, Kim; Iruthayaraj, Joseph

2013-08-15

This work demonstrates the application of carbohydrate based methacrylate polymer brush, poly(2-lactobionamidoethyl methacrylate), for the purpose of cell adhesion studies. The first part of the work illustrates the effects of the structure of the aminosilane based ATRP initiator layer on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass substrate. It is suggested that the interaction of the galactose units of the polymer brush with the asialoglycoprotein receptor (ASGPR) of HepG2 cells has resulted in the protein enrichment along the cell periphery. Copyright © 2013 Elsevier Inc. All rights reserved.

The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

PubMed

Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

2015-01-15

Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

[Anti-proliferation Effect of Taraxacum mongolicum Extract in HepG2 Cells and Its Mechanism].

PubMed

Guo, Jun-bin; Ye, Hai-hong; Chen, Jian-feng

2015-10-01

To study the anti-proliferation effect of Taraxacum mongolicum extract in HepG2 cells and its mechanism. The total proteins of HepG2 cells treated with Taraxacum mongolicum extract were. extracted and mitochondria-mediated apoptosis-related proteins (Survivin, Mcl-1, BCL-xL, BCL-2, Smac, BAX, Bad, Cytochrome c and Caspase-3/7/9) were detected by Western blot. Taraxacum mongolicum extract obviously inhibited the proliferation of HepG2 cells and the expression of anti-apoptotic proteins (Survivin, BCL-xL and BCL-2), increased the expression of pro-apoptotic proteins (Smac and Caspase-3/7/9), and promoted the release of Cytochrome c from mitochondria to cytoplasm in HepG2 cells. The effects were in a dose-independent mode. Taraxacum mongolicum extract can inhibit the proliferation of HepG2 cells and the anti-proliferation mechanism is related to mitochondria-mediated apoptosis.

In vitro synthesis and processing of herpes simplex virus type 2 gG-2, using cell-free transcription and translation systems.

PubMed Central

Weldon, S K; Su, H K; Fetherston, J D; Courtney, R J

1990-01-01

Translation of in vitro-synthesized herpes simplex virus type 2 (HSV-2) gG-2 mRNA in a reticulocyte lysate system was used to study the processing of HSV-2 gG-2. In the presence of canine pancreatic microsomal membranes, a single species that is protected from trypsin digestion was detected. This product comigrates with the 104,000-Mr (104K) high mannose intermediate seen in HSV-2-infected-cell lysates. Endo-beta-N-acetylglucosaminidase H treatment of the in vitro-synthesized 104K protein yielded a single product migrating at 100 K. The 72K and 31K cleavage products of gG-2 were not observed in the in vitro system. These data show that the molecular weight of the nonglycosylated form of the gG-2 protein is 100,000 and that the cotranslational processing of this protein in the endoplasmic reticulum yields the 104K high-mannose intermediate. Images PMID:2154614

Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

EPA Science Inventory

Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

Identification of acetylshikonin as the novel CYP2J2 inhibitor with anti-cancer activity in HepG2 cells.

PubMed

Park, See-Hyoung; Phuc, Nguyen Minh; Lee, Jongsung; Wu, Zhexue; Kim, Jieun; Kim, Hyunkyoung; Kim, Nam Doo; Lee, Taeho; Song, Kyung-Sik; Liu, Kwang-Hyeon

2017-01-15

Acetylshikonin is one of the biologically active compounds derived from the root of Lithospermum erythrorhizon, a medicinal plant with anti-cancer and anti-inflammation activity. Although there have been a few previous reports demonstrating that acetylshikonin exerts anti-cancer activity in vitro and in vivo, it is still not clear what is the exact molecular target protein of acetylshikonin in cancer cells. The purpose of this study is to evaluate the inhibitory effect of acetylshikonin against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. The inhibitory effect of acetylshikonin on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its cytotoxicity against human hepatoma HepG2 cells was also evaluated. Astemizole, a representative CYP2J2 probe substrate, was incubated in HLMs in the presence or absence of acetylshikonin. After incubation, the samples were analyzed by liquid chromatography and triple quadrupole mass spectrometry. The anti-cancer activity of acetylshikonin was evaluated on human hepatocellular carcinoma HepG2 cells. WST-1, cell counting, and colony formation assays were further adopted for the estimation of the growth rate of HepG2 cells treated with acetylshikonin. Acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylation activity (K i = 2.1µM) in a noncompetitive manner. The noncompetitive inhibitory effect of acetylshikonin on CYP2J2 enzyme was also demonstrated using this 3D structure, which showed different binding location of astemizole and acetylshikonin in CYP2J2 model. It showed cytotoxic effects against human hepatoma HepG2 cells (IC 50 = 2μM). In addition, acetylshikonin treatment inhibited growth of human hepatocellular carcinoma HepG2 cells leading to apoptosis accompanied with p53, bax, and caspase3 activation as well as bcl2 down-regulation. Taken together, our present study elucidates acetylshikonin displays the

Withaferin A inhibits the proliferation of gastric cancer cells by inducing G2/M cell cycle arrest and apoptosis.

PubMed

Kim, Green; Kim, Tae-Hyoun; Hwang, Eun-Ha; Chang, Kyu-Tae; Hong, Jung Joo; Park, Jong-Hwan

2017-07-01

Human gastric adenocarcinoma (AGS) is one of the most common types of malignant tumor and the third-leading cause of tumor-associated mortality worldwide. Withaferin A (WA), a steroidal lactone derived from Withania somnifera , exhibits antitumor activity in a variety of cancer models. However, to the best of our knowledge, the direct effect of WA on AGS cells has not previously been determined. The present study investigated the effects of WA on the proliferation and metastatic activity of AGS cells. WA exerted a dose-dependent cytotoxic effect on AGS cells. The effect was associated with cell cycle arrest at the G2/M phase and the expression of apoptotic proteins. Additionally, WA treatment resulted in a decrease in the migration and invasion ability of the AGS cells, as demonstrated using a wound healing assay and a Boyden chamber assay. These results indicate that WA directly inhibits the proliferation and metastatic activity of gastric cancer cells, and suggest that WA may be developed as a drug for the treatment of gastric cancer.

Withaferin A inhibits the proliferation of gastric cancer cells by inducing G2/M cell cycle arrest and apoptosis

PubMed Central

Kim, Green; Kim, Tae-Hyoun; Hwang, Eun-Ha; Chang, Kyu-Tae; Hong, Jung Joo; Park, Jong-Hwan

2017-01-01

Human gastric adenocarcinoma (AGS) is one of the most common types of malignant tumor and the third-leading cause of tumor-associated mortality worldwide. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, exhibits antitumor activity in a variety of cancer models. However, to the best of our knowledge, the direct effect of WA on AGS cells has not previously been determined. The present study investigated the effects of WA on the proliferation and metastatic activity of AGS cells. WA exerted a dose-dependent cytotoxic effect on AGS cells. The effect was associated with cell cycle arrest at the G2/M phase and the expression of apoptotic proteins. Additionally, WA treatment resulted in a decrease in the migration and invasion ability of the AGS cells, as demonstrated using a wound healing assay and a Boyden chamber assay. These results indicate that WA directly inhibits the proliferation and metastatic activity of gastric cancer cells, and suggest that WA may be developed as a drug for the treatment of gastric cancer. PMID:28693185

Anti-Colon Cancer Effects of 6-Shogaol Through G2/M Cell Cycle Arrest by p53/p21-cdc2/cdc25A Crosstalk.

PubMed

Qi, Lian-Wen; Zhang, Zhiyu; Zhang, Chun-Feng; Anderson, Samantha; Liu, Qun; Yuan, Chun-Su; Wang, Chong-Zhi

2015-01-01

Chemopreventive agents can be identified from botanicals. Recently, there has been strong support for the potential of 6-shogaol, a natural compound from dietary ginger (Zingiber officinale), in cancer chemoprevention. However, whether 6-shogaol inhibits the growth of colorectal tumors in vivo remains unknown, and the underlying anticancer mechanisms have not been well characterized. In this work, we observed that 6-shogaol (15 mg/kg) significantly inhibited colorectal tumor growth in a xenograft mouse model. We show that 6-shogaol inhibited HCT-116 and SW-480 cell proliferation with IC50 of 7.5 and 10 μM, respectively. Growth of HCT-116 cells was arrested at the G2/M phase of the cell cycle, primarily mediated by the up-regulation of p53, the CDK inhibitor p21(waf1/cip1) and GADD45α, and by the down-regulation of cdc2 and cdc25A. Using p53(-/-) and p53(+/+) HCT-116 cells, we confirmed that p53/p21 was the main pathway that contributed to the G2/M cell cycle arrest by 6-shogaol. 6-Shogaol induced apoptosis, mainly through the mitochondrial pathway, and the bcl-2 family might act as a key regulator. Our results demonstrated that 6-shogaol induces cancer cell death by inducing G2/M cell cycle arrest and apoptosis. 6-Shogaol could be an active natural product in colon cancer chemoprevention.

MyoD undergoes a distinct G2/M-specific regulation in muscle cells.

PubMed

Batonnet-Pichon, Sabrina; Tintignac, Lionel J; Castro, Anna; Sirri, Valentina; Leibovitch, Marie Pierre; Lorca, Thierry; Leibovitch, Serge A

2006-12-10

The transcription factors MyoD and Myf5 present distinct patterns of expression during cell cycle progression and development. In contrast to the mitosis-specific disappearance of Myf5, which requires a D-box-like motif overlapping the basic domain, here we describe a stable and inactive mitotic form of MyoD phosphorylated on its serine 5 and serine 200 residues by cyclin B-cdc2. In mitosis, these modifications are required for releasing MyoD from condensed chromosomes and inhibiting its DNA-binding and transcriptional activation ability. Then, nuclear MyoD regains instability in the beginning of G1 phase due to rapid dephosphorylation events. Moreover, a non-phosphorylable MyoD S5A/S200A is not excluded from condensed chromatin and alters mitotic progression with apparent abnormalities. Thus, the drop of MyoD below a threshold level and its displacement from the mitotic chromatin could present another window in the cell cycle for resetting the myogenic transcriptional program and to maintain the myogenic determination of the proliferating cells.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

PubMed

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

PubMed

Shi, Hong-Bo; Sun, Hai-Qing; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Chen, De-Xi; Lou, Jin-Li; Duan, Zhong-Ping

2015-05-07

To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.