Geranium thunbergii extract-induced G1 phase cell cycle arrest and apoptosis in gastric cancer cells

ABSTRACT

Geranium thunbergii is a traditional East Asian medicine for stomach diseases including dysentery and stomach ulcers in East Asia and has been reported to possess biological activity. The benefits of G. thunbergii in gastric cancer are unknown. In this study, we demonstrate that G. thunbergii extract suppresses proliferation and induces death and G1/S cell cycle arrest of gastric cancer cells. Proliferation was significantly inhibited in a time- and dose-dependent manner. Cell cycle arrest was associated with significant decreases in CDK4/cyclinD1 complex and CDK2/cyclinE complex genes expression. In addition, the protein expression of caspase-3 was decreased and that of activated poly (ADP-ribose) polymerase (PARP) was increased, which indicated apoptosis. The expressions of the Bax and Bcl-2, which are apoptosis related proteins, were upregulated and down-regulated, respectively. The results indicate that G. thunbergii extract can inhibit proliferation and induce both G/S cell cycle arrest and apoptosis of gastric cancer cells. Also, the induction of apoptosis involved the intrinsic pathways of the cells. Take the results, we suggest that G. thunbergii extract has anti-gastric cancer activity and may be a potential therapeutic candidate for gastric cancer.     

Overexpression of WDR79 in non‐small cell lung cancer is linked to tumour progression

WD‐repeat protein 79 (WDR79), a member of the WD‐repeat protein family, acts as a scaffold protein, participating in telomerase assembly, Cajal body formation and DNA double‐strand break repair. Here, we first report that WDR79 is frequently overexpressed in cell lines and tissues derived from non‐small cell lung cancer (NSCLC). Knockdown of WDR79 significantly inhibited the proliferation of NSCLC cells in vitro and in vivo by inducing cell cycle arrest and apoptosis. WD‐repeat protein 79 ‐induced cell cycle arrest at the G0/G1 phase was associated with the expression of G0/G1‐related cyclins and cyclin‐dependent kinase complexes. We also provide evidence that WDR79 knockdown induces apoptosis via a mitochondrial pathway. Collectively, these results suggest that WDR79 is involved in the tumorigenesis of NSCLC and is a potential novel diagnostic marker and therapeutic target for NSCLC.     

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3′-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.     

CDK1 siRNA干扰在Taxol诱导细胞凋亡中的作用

目的研究转染细胞周期依赖性蛋白激酶1（cyclin．dependent kinase1,CDK1）siRNA、以及转染后进行凋亡刺激对细胞周期和凋亡的影响,探讨CDK1在细胞凋亡中的确切作用,揭示细胞周期与细胞凋亡协调的分子机制。方法以人宫颈癌细胞株HeLa细胞为研究对象,脂质体转染CDK1siRNA,转染后48h加紫杉醇（Tax01）（20μg／m1）刺激凋亡,Western印迹检测CDK1和抗凋亡蛋白BCL2表达,AnnexinV／PI法检测细胞的凋亡,流式细胞仪分析DNA含量检测细胞周期。结果转染CDK1 siRNA后,CDK1蛋白的表达下降,细胞周期G2／M期比例增加,细胞凋亡率与对照相比没有明显升高。只加Taxol刺激12h后细胞凋亡率增加并伴有S期和G2／M期比例增加。转染CDKlsiRNA后再用Taxol刺激,其细胞凋亡率没有明显改变,G2／M期阻滞效应也没有叠加。BCL2蛋白只在加Taxol刺激组表达下降,与CDK1表达减少没有相关性。结论siRNA沉默导致的CDK1表达降低只导致细胞周期G2／M期阻滞,没有引起细胞凋亡;CDK1的表达降低对紫杉醇所诱导的细胞周期阻滞和细胞凋亡效应没有明显影响。     

Increased levels of the cell cycle inhibitor protein, dacapo, accompany 20-hydroxyecdysone-induced G1 arrest in a mosquito cell line

When treated with the steroid hormone 20‐hydroxyecdysone (20E), C7‐10 cells from the mosquito, Aedes albopictus, arrest in the G1 phase of the cell cycle. To explore whether 20E‐mediated cell cycle arrest proceeds through increased levels of cell cycle inhibitor (CKI) proteins, we cloned the Ae. albopictus homolog of dacapo, the single member of the Cip/Kip family of CKI proteins known from Drosophila melanogaster. The Ae. albopictus dacapo cDNA encoded a 261‐amino acid homolog of the Aedes aegypti protein XP_001651102.1, which is encoded by an ～23 kb gene containing three exons. Like dacapo from D. melanogaster, the ～27 kDa protein from Aedes and Culex mosquitoes contained several S/TXXE/D motifs corresponding to potential protein kinase CK2 phosphorylation sites, and a binding site for proliferating cell nuclear antigen (PCNA). When extracts from cells treated with 20E were analyzed by western blotting, using a primary antibody to synthetic peptides from the mosquito dacapo protein, up‐regulation of an ～27 kDa protein was observed within 24 h, and the abundance of the protein further increased by 48 h after hormone treatment. This is the first investigation of a cell cycle inhibitory protein in mosquitoes. The results reinforce growing evidence that 20E affects expression of proteins that regulate cell cycle progression. © 2011 Wiley Periodicals, Inc.     

Antiproliferative Activity of Chemically Characterized Propolis from Turkey and Its Mechanisms of Action

The aim of this study was to evaluate the ethanolic extract of propolis originated from northern Turkey for its antiproliferative, apoptotic and cell cycle arrest promoting effects on MCF7, HGC27, A549 cancer cell lines and a healthy cell line (HUVEC) in terms of DNA content, morphological features, expression of cell cycle checkpoint proteins p21, p53, Cyclin D1 and immune checkpoint protein PD‐L1. The extract showed moderate antiproliferative activity against all tested cancer cell lines with IC₅₀ values in the range of 58.6–90.7 μg/mL in MTS assay. Further studies indicated that propolis extract exerted apoptotic effect on cancer cell lines, promoted cell cycle arrest through activation of p21 and resulted in accumulation at G0/G1 phase of cancer cells. Propolis treatment caused increased cell size, according to fluorescent imaging except for MCF7. HPTLC analysis revealed that 3‐O‐methylquercetin, chrysin, caffeic acid, CAPE, galangin and pinocembrin were the main components of the extract. The amounts of caffeic acid and CAPE in the extract were found to be 5.5 and 11.1 mg/g, respectively, by a validated HPLC method. Our study is the first one, revealing effect of propolis on PD‐L1 expression on certain cancer cell lines.     

The caveolin‐3 P104L mutation in LGMD‐1C patients inhibits non‐insulin‐stimulated glucose metabolism and growth but promotes myocyte proliferation

The caveolin‐3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb‐girdle muscular dystrophy type 1C (LGMD‐1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3‐P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p‐GSK3β, and p‐p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3‐P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD‐1C.     

转录因子HBP1抑制骨肉瘤细胞增殖

转录因子HBP1(HMG-box containing protein 1, HBP1)属HMG家族,是1个含有513个氨基酸残基的多肽,可与RB蛋白结合,抑制许多癌基因的表达,从而抑制细胞的增殖. 本工作构建了HBP1慢病毒表达载体,病毒包装后感染骨肉瘤细胞U2OS细胞.Western印迹结果显示,感染细胞cyclin D1、c-Myc蛋白质水平降低 ,而p53蛋白质水平增加;Real-time PCR检测cyclin D1、c-Myc及p53 mRNA水平与蛋白质检测结果一致. HBP1表达载体（pEFBOS-HBP1）和报告基因载体（含 cyclin D1、c-Myc或p53 promoter）共转染U2OS细胞后的荧光素酶分析发现,HBP1可抑制cyclin D1、c-Myc启动子转录激活,促进p53启动子转录激活,并且这种抑制或激活作用具有HBP1剂量依赖性. 以细胞代龄群体倍增值PD （population doubling）为指标测试细胞生长结合软琼脂集落形成实验证明,HBP1慢病毒感染的U2OS细胞生长速度减缓、集落形成能力下降. 上述结果提示,HBP1可能通过调控细胞增殖相关基因的表达抑制骨肉瘤细胞的增殖.     

Induction of mitotic catastrophe by PKC inhibition in Nf1-deficient cells

Mutations of tumor suppressor Nf1 gene deregulate Ras-mediated signaling, which confers the predisposition for developing benign or malignant tumors. Inhibition of protein kinase C (PKC) was shown to be in synergy with aberrant Ras for the induction of apoptosis in various types of cancer cells. However, it has not been investigated whether loss of PKC is lethal for Nf1-deficient cells. In this study, using HMG (3-hydroxy-3-methylgutaryl, a PKC inhibitor), we demonstrate that the inhibition of PKC by HMG treatment triggered a persistently mitotic arrest, resulting in the occurrence of mitotic catastrophe in Nf1-deficient ST8814 cells. However, the introduction of the Nf1 effective domain gene into ST8814 cells abolished this mitotic crisis. In addition, HMG injection significantly attenuated the growth of the xenografted ST8814 tumors. Moreover, Chk1 was phosphorylated, accompanied with the persistent increase of cyclin B1 expression in HMG-treated ST8814 cells. The knockdown of Chk1 by the siRNA prevented the Nf1-deficient cells from undergoing HMG-mediated mitotic arrest as well as mitotic catastrophe. Thus, our data suggested that the suppression of PKC activates the Chk1-mediated mitotic exit checkpoint in Nf1-deficient cells, leading to the induction of apoptosis via mitotic catastrophe. Collectively, the study indicates that targeting PKC may be a potential option for developing new strategies to treat Nf1-deficiency-related diseases.     

Arsenic trioxide induces G2/M arrest in hepatocellular carcinoma cells by increasing the tumor suppressor PTEN expression

Arsenic trioxide (As₂O₃), an effective agent against acute promyelocytic leukemia, has been reported to inhibit the viability of solid tumors cell lines recently. The detailed molecular mechanism underlying the As₂O₃‐induced inactivation of the cdc2 and possible functional role of PTEN in the observed G2/M arrest has yet to be elucidated. Here, we assessed the role of PTEN in regulation of As₂O₃‐mediated G2/M cell cycle arrest in Hepatocellular carcinoma cell lines (HepG2 and SMMC7721). After 24 h following treatment, As₂O₃ induced a concentration‐dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by As₂O₃ is associated with decreased cdc2 protein and increased phospho‐cdc2(Tyr15). As₂O₃ treatment increased Wee1 levels and decreased phospho‐Wee1(642). Moreover, As₂O₃ substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in As₂O₃‐treated cells increased phospho‐Wee1(Ser642) while decreased phospho‐cdc2(Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by As₂O₃ involved upregulation of PTEN. J. Cell. Biochem. 113: 3528–3535, 2012. © 2012 Wiley Periodicals, Inc.     

Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast

The START cell cycle transition in the budding yeast Saccharomyces cerevisiae is catalyzed by the Cdc28 cyclin-dependent kinase associated with Cln-type cyclins. Since ectopic expression of the B-type cyclin CLB5 can efficiently rescue the inviability that results from CLN depletion, we tested the specificity of the CLN and CLB classes of cyclins for promoting START-associated events. Several aspects of the regulation of the mating factor response were compared for cells in which START activity was provided by either Cln-cyclins or Clb5. Unlike Cln1 and Cln2, high level expression of Clb5 was unable to repress the activity of the mating factor response pathway at START. Downregulation of Far1 protein at START is normal in cln−GAL1::CLB5 cells. Even though the Clb5-Cdc28 kinase activity in cln−GAL1::CLB5 cells is not downregulated in response to mating factor, cells arrest in the first cycle after addition of mating factor with a similar sensitivity as wild-type cells. However, whereas wild-type cells treated with mating factor arrest specifically in G1 phase as unbudded cells with unreplicated DNA (pre-START), most cln−GAL1::CLB5 cells arrest as budded post-START cells with replicated DNA. Our findings demonstrate the ability of post-START cells to arrest in response to mating factor and provide novel evidence for mechanisms that contribute to restrict mating factor-induced arrest in wild-type cells to the G1 phase of the cell cycle. Received: 25 September 1997 / Accepted: 9 November 1997     

Functional Analysis of the Thioredoxin Domain in Porphyromonas gingivalis HBP35

Periodontitis is one of the most common oral diseases in humans. This caused by infection by the oral bacterium Porphyromonas gingivalis. Our strategy to prevent this infection is to establish a passive immunization system in which endogenous antibodies can be applied directly to neutralize virulent factors associated with this bacterium. We focused our attention on the P. gingivalis 35 kDa surface protein, or HBP35, since this protein is involved not only in the coaggregation with oral miroflora but also in hemin binding. In addition, nucleotide sequencing of the gene, hbp35, coding for this protein revealed the presence of a catalytic center for thioredoxin, and we further attempted to characterized the protein by amino acid substitution. A total of four Cys residues were substituted for Ser residues by combining the simple method for site-directed mutagenesis and the heterodimer system, an approach designed to construct chimeric plasmids readily. Native and mutagenized hbp35 were introduced into the Eschericha coli dsbA mutant strain, JCB 572, defective in both alkaline phosphatase and motile activities due to inefficient disulfide bond formation. Transformant harboring the native hbp35 could complement the dsbA mutation, suggesting a role of disulfide bond formation of this protein in P. gingivalis cells. Possible roles of the Cys residues in complementation are discussed.     

ECRG1, a novel candidate of tumor suppressor gene in the esophageal carcinoma, triggers a senescent program in NIH3T3 cells

Esophageal cancer-related gene 1 (ECRG1) is a novel tumor-suppressor gene candidate identified from the human esophagus. Previous studies showed that ECRG1 overexpression could inhibit cell growth and induce G1 cell cycle arrest and p15(INK4b) expression by interacting with Miz-1 (Myc-interacting zinc finger protein). Such evidence suggests the alterations in ECRG1 may play an important role in tumorigenesis. To further study the biological function of the ECRG1 gene, we transfected ECRG1 into NIH3T3 cells. Expression of ECRG1 in these cells caused senescence-like changes characterized in terms of altered cell morphology, cell cycle arrest at the G1/S phase, and significantly impaired cell proliferation (P < 0.01). Moreover, NIH3T3 cells transfected with ECRG1 stained positive for SA-beta-gal staining (pH 6.0), which is a specific marker of cellular senescence. We also studied changes in telomerase activity and the related senescence genes, such as p21 and p16. The results indicated that when ECRG1 induced a senescence-like state, telomerase activity was markedly decreased (P < 0.05), and expression of p21 was distinctly increased, whereas no changes were detected in p16 and telomerase-component RNA levels. These findings suggest that ECRG1 may be of importance in murine cell senescence, promoting senescence by regulating expression of p21.     

Identification of Vaccinia‐H1 Related Phosphatase as an Anticancer Target for 1,2,3,4,6‐O‐Pentagalloylglucose

Protein tyrosine phosphatases are involved in diverse human diseases, including cancer, diabetes and inflammatory disorders. Loss of Vaccinia‐H1 related phosphatase (VHR) has been shown to arrest at the G1‐S and G2‐M transitions of the cell cycle, and to increases cell death of prostate cancer cells through JNK activation, suggesting that VHR can be considered as an anticancer target. In this study, 658 natural products were screened through in vitro enzyme assay to identify VHR inhibitor. Among the VHR‐inhibitory compounds, 1,2,3,4,6‐O‐pentagalloylglucose (PGG) was selected for further study as it has been reported to show antitumor effects against tumor model mice, but its direct target has not been identified. PGG inhibited the catalytic activity of VHR (K_i=53 nm ) in vitro. Furthermore, the incubation of HeLa cervical cancer cells with PGG dramatically decreased cell viability and markedly increased the protein levels of the cleaved PARP, a hallmark of apoptosis. In addition, treatment of HeLa cells with PGG significantly reduced the protein levels of cyclin D1, Bcl‐2 and STAT3 phosphorylation. Taken together, these results suggest that PGG could be a potential therapeutic candidate for the treatment of cervical cancer through VHR inhibition.