Z-Guggulsterone alleviated renal fibrosis and G_(2)/M cycle arrest through Klotho/P53 signaling

OBJECTIVE Chronic kidney disease(CKD)has become a global public health problem with 10%-15%incidence rate,and inhibiting the renal interstitial fibrosis is considered to be a potential strategy to delay the progression of CKD.Z-Guggulsterone(Z-GS),an active compound from derived from Commiphora mukul,has been proved to be effective in various diseases.The present study aimes to determine the protective effect and the molecular mechanism of Z-GS on renal fibrosis.METHODS Unilateral ureteral obstruction(UUO)mice and hypoxia-induced HK-2 cells were used to simulate renal fibrosis in vitro and in vivo,respectively.The mice and cells were treated with different doses of Z-GS to observe the pharmacological action.Renal function,including Scr,BUN,and UA,were detected by commercial kits.H&E and Masson staining were performed to observe histopathological changes of kidney.Cell viability and LDH release of HK-2 cells were detected by commercial kits.Cell cycle distribution and apoptosis rate were analyzed by flow cytometry.Fibrosis markers were detected by immunohistochemistry and immunofluorescence analysis.Cell cycle related proteins and Klotho/p53 signaling were analyzed by Western blotting.RESULTS The results showed that Z-GS decreased the rise of Scr,BUN,and UA and lightened renal histopathological injury,which were induced by UUO.Besides,Z-GS administration alleviated renal fibrosis in mice by inhibiting the expressions ofα-SMA,TGF-βand collagenⅣ,and delayed G2/M cell cycle arrest by promoting the expressions of CDK1 and cyclinD1/B1 rate.Experiments in vitro indicated that Z-GS treatment significantly increased the cell viability while decreased the LDH release in hypoxia-induced HK-2 cells.In addition,hypoxia induced fibrosis and G2/M cycle arrest in HK-2 cells were retarded by Z-GS.The study of its possible mechanism exhibited that Z-GS treatment increased the level of Klotho and inhibited P53 level.Nevertheless,the effect of Z-GS on Klotho/P53 signaling was reversed by siRNA-Klotho.Moreover,siRNA-Klotho treatment eliminated the effects of Z-GS on G2/M cell cycle arrest and fibrosis.CONCLUSION This study clarified that Z-GS alleviated renal fibrosis and G2/M cycle arrest through Klotho/P53 signaling pathway.People who have suffered CKD may potentially benefit from treatment with Z-GS.

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

Objective： To examine the apoptotic effect of ent-llα-hydroxy-15-oxo-kaur-16-en-19-oic-acid （5F）, a compound isolated from Pteris semipinnata L （PsL）, in human lung cancer A549 cells. Methods： A549 cells were treated with 5F （0-80 lag/ml） for different time periods. Cytotoxicity was examined using a Ml-I- method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay （ELISA） and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species （ROS） level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results： 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor （AIF）, and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion： 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.

Viral infections and cell cycle G2/M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast (Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well- characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest. Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

Research on cell cycle retardation, apoptosis and the expression of antioncogene p57^(Kip2) by radioactive rays in nasopharyngeal carcinoma cell line in vitro

Objective: To study cell cycle retardation, apoptosis and the expression of antioncogene p57kip2 by radioactive rays in nasopharyngeal carcinoma cells. Methods: Cell cycle retardation, apoptosis and cell survival rate induced by radioac- tive rays were tested by the methods of flow cytometry and MTT method. The expression of antioncogene p57kip2 was detected by immunohistochemistry and Western blot. Results: After irradiation, G1 phase had no obvious retardation, S phase showed transient delay. There was a positive correlation between irradiation dosage and retardation strength in G2/M phase (P < 0.01). Peak value appeared at 24 h after 12 Gy irradiation, then decreased. There was a positive correlation between apop- tosis incidence and irradiation dosage or after-irradiation time extention (P < 0.01). There was a negative correlation between cell survival rate and irradiation dosage or apoptosis incidence (P < 0.01). The expression of p57kip2 protein was up-regulated along with the prolongation of time and dosage after irradiation (P < 0.01). Conclusion: G2/M phase arrest, apoptosis and the up-regulation of the expression of p57kip2 protein all can reflect predict the radiosensitivity of nasopharyngeal carcinoma cells.

Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As 2O 3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As 2O 3 induced cell apoptosis, K562 cells were cultured with As 2O 3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As 2O 3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G 2/M phase increased in proportion to As 2O 3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As 2O 3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G 2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As 2O 3-induced apoptosis.

Cucurbitacin E inhibits the proliferation of hepatoma cells in vitro and in vivo through induction of G2/M phase arrest

Objective Cucurbitacins are the highly oxygenated tetracyclic triterpenes,which are predominantly found in the Cucurbitaceae family but are also present in several other families of the plant kingdom.A number of compounds of this group have been investigated for their cytotoxic,hepatoprotective,anti-inflammatory,cardiovascular and anti-diabetic activities.In China,the cucurbitacin preparation,which contains mostly cucurbitacin B and cucurbitacin E,has been clinically used for the treatment of the primary liver carcinoma.It has been previously reported that cucurbitacin E could produce cytotoxicity against a variety of cancer cells,and various mechanisms were implicated in its cytotoxic effect.The present study is to investigate the effect of cucurbitacin E on hepatoma cells in vitro and in vivo and to study their potential mechanisms of action.Methods The MTT assay was used to assess the viability of human HepG2 and BEL7402 hepatoma cells in vitro after treatment with different concentrations of cucurbitacin E.The cell cycle distribution was determined by flowcytometric analysis after propidium iodide(PI)staining.The cell cycle-related proteins were detected using western blotting analysis.Implanted mouse hepatoma H22 model was built to evaluate the growth inhibitory effect of cucurbitacin E in vivo in mice.Results Our studies found that cucurbitacin E(10-300 nM)produced anti-proliferative effect on human HepG2 and BEL7402 hepatoma cells in vitro without cytotoxicity.According to flowcytometric analysis,cucurbitacin E arrested the cell cycle at G2/M phase in both HepG2 and BEL7402 hepatoma cells after 24 h treatment.Cucurbitacin E induced the decrease in the level of CDK1 protein and the increase in the level of p21 protein,but had no effect on the levels of cyclin A,cyclin B1 and Cdc25C protein.In in vivo anti-tumor experiment,cucurbitacin E had significant inhibitory effects on the growth of mouse H22 hepatoma cells.Conclusions Cucurbitacin E inhibited the proliferation of hepatoma cells in vitro and in vivo,at least in part,through induction of cell cycle arrest at G2/M phase,which was mediated by concomitant upregulation of p21 and downregulation of CDK1.We consider that cucurbitacin E may be useful in the treatment of liver cancer.

2(±)-7,8,3',4',5'-Pentamethoxyflavan induces G2/M phase arrest and apoptosis in human leukemia HL60 cells

Objective Flavans are a set of naturally occurring flavonoids possessing a 2-phenylchroman nucleus,which are widely distributed in the plant kingdom.A number of flavan compounds exhibit antitumor activities.In our previous report,a straightforward synthetic procedure for 2(±)-7,8,3',4',5'-pentamethoxyflavan(PMF)was developed.To be more important,PMF showed growth inhibitory effect on various human tumor cell lines,especially against HL60 cells.In the present study,we aim to investigate the molecular mechanisms of action of PMF in HL60 cells.This is the first report of the molecular mechanisms on anti-tumor effect of flavan compounds.Methods Trypan blue exclusion experiment was used for cell growth inhibition assay.Cell apoptosis,cell cycle distribution and the mitochondrial membrane potential(MMP)were assessed by flowcytometric analysis after AO/EB,PI and Rh123 flurescence staining,respectively.Cell cycle-and apoptosis-related proteins were detected using western blotting analysis.Results PMF(1-30 μM)inhibited the growth of HL60 cells in a time-and concentration-dependent manner.Antiproliferative effect of PMF on HL60 cells was associated with G2/M cell cycle arrest,which was mediated by regulating the expression of p21,Cdc25C and cyclin A proteins and inhibiting the phosphorylation of Cdc2 at Thr161.The prolonged PMF treatment also induced apoptosis of HL60 cells,which was characterized by DNA fragmentation,cleavage of poly(ADP-ribose)polymerase,caspase-3,caspase-8 and caspase-9,changes of Bcl-2 and Bax expression and a decrease in the mitochondrial membrane potential(MMP).Furthermore,caspase-3 inhibitor,not caspase-8 inhibitor and caspase-9 inhibitor,completely blocked PMF-caused apoptosis.Conclusions PMF inhibited the growth of HL60 cells via induction of G2/M arrest and apoptosis.Blockade of cell cycle was associated with the downregulation of Cdc2 complex activity.Both death receptor and mitochondrial apoptotic pathways explained PMF-caused apoptosis.

A RAPID ESTIMATION OF CELL CYCLE PARAMETERS：ONE HOUR COUNTINUOUS BROMODEOXYURIDINE（BrdUrd）LABELING METHOD

For any cultured cell lines during their exponentially growing phase,the outflow rate of G2M cells can be measured with bromodeoxyuridine (BrdUrd) continuous labeling.The duration of G2M phase can be estibmated from the slope of the outtflow rate, The cell cycling time and the durations of G1 phase and S phase can be calculated from the measured fractions of G1, S cells and the cell age distribution theory. The method described here was simple and only about 1 hour was needed Several cell lines were measured and the obtained results were equivalent to the values determined by the FLSm curve or other methods.

金雀异黄素抑制人胃癌细胞增殖与G_2/M期阻滞作用的体外研究

目的 : 研究金雀异黄素 (genistein,Gen)对人胃癌 SGC-790 1细胞增殖抑制作用及对细胞周期的影响。方法 : 采用3H-Td R掺入液闪计数法观察 Gen对人胃癌细胞增殖影响 ,流式细胞仪分析细胞周期分布 ,免疫组化和 Western Blotting分别检测 cyclin B、P2 1 waf1 / cip1 蛋白表达情况。结果 : Gen对胃癌细胞生长有显著抑制作用 ,使细胞生长停滞于 G2 / M期 ,并使细胞cyclin B、P2 1 waf1 / cip1蛋白表达增加 ,且呈剂量 -效应关系。结论 : Gen在此剂量下抑制胃癌细胞增殖、诱导 G2 / M期阻滞与其稳定 cyclin B蛋白和上调 P2 1 waf1 / cip1

E1/E3缺失型腺病毒载体引起细胞周期G_2/M阻滞

腺病毒载体广泛应用于基因治疗和转基因研究 ,目前常用的E1 E3缺失型复制缺陷腺病毒载体虽然失去了病毒复制必需的E1基因 ,但载体上的其它病毒基因仍能在宿主细胞内表达 .为研究这些基因对细胞的毒性作用 ,选择了 3种携带没有明显细胞毒性外源基因的腺病毒载体 ,观察感染 2种肿瘤细胞后细胞核形态改变 ,并用流式细胞仪检测细胞周期及凋亡情况 .发现大剂量重组缺陷型腺病毒感染细胞后引起细胞变圆 ,核增大 ,细胞周期阻滞于G2 M期 ,继而染色质凝聚 ,细胞发生坏死或凋亡 ;各种腺病毒载体造成G2 M阻滞所需感染量不同 ,但都随时间延长和感染量增加而加重 .这些结果提示腺病毒基因对细胞的影响是多方面的 ,在以此类病毒载体进行基因转移和基因治疗的研究中 ,精确滴定病毒滴度和转导效率非常重要 。

肾小管上皮细胞G2-M期阻滞在缺氧诱导的肾间质纤维化中的作用

目的:探讨肾小管上皮细胞G2-M期阻滞在缺氧诱导的肾间质纤维化中的作用。方法:体外实验,人肾小管上皮细胞(HK2)分别置于常氧21%和缺氧1%的孵箱里,培养24,48 hrs。采用流式细胞术检测缺氧48 hrs后上皮细胞的周期分布;采用Western blot法检测Collagen4A1(COL4A1)及α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平。单侧输尿管梗阻(UUO)14 d小鼠模型,HE及Masson染色观察肾组织病理改变及纤维化程度,免疫组化染色观察α-SMA的表达及部位;Western blot检测低氧标记分子HIF-1α、细胞周期蛋白cyclinB1和cyclinD1的蛋白水平。结果:与常氧组比较,缺氧组肾小管上皮细胞G2/M期比例增高(P<0.05),同时α-SMA和COL4A1的蛋白表达水平增高(P<0.05);与假手术组比较,HE及Masson染色显示模型组肾组织纤维化程度增高,α-SMA的表达增高且主要分布在间质中;Western blot结果显示:HIF-1α及G2/M期标记物cyclinB1/cyclinD1比值增加。结论:肾小管上皮细胞G2-M期阻滞参与缺氧诱导的肾间质纤维化。

p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis

Objective To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest.Methods Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation.Depletion of p21 was carried out by employing the siR NA technique.Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28,an M-phase marker.Senescence was assessed by senescenceassociated-β-galactosidase（SA-β-gal） staining combined with Ki67 staining,a cell proliferation marker.Results Accompanying increased p21,the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays.Furthermore,these irradiated cells were blocked at the G2 phase followed by cellular senescence.Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases,as well as the high expression of histone H3 phosphorylated at Ser28.Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells.However,cells with serious DNA damage failed to undergo cytokinesis,leading to the accumulation of multinucleated cells.Conclusion Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation.Downregulation of p21 by siR NA resulted in G2-arrested cells entering into mitosis with serious DNA damage.This is the first report on elucidating the role of p21 in the bypass of mitosis.

Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20mel Rather Than DNA Demethylation

Objective:To investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia(AL)cells.Methods:The expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines.Pyrophosphate sequencing was performed to determine the methylation degree.Then,the enrichment of H4K20mel and H3K9ac was determined using ChIP-qPCR.Flow cytometry was used to analyze the cell cycle.Results:The IC_(50) of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line.Genistein upregulated H4K20mel,KMT5A and Wnt suppressor genes,including Wnt5a,and downregulated the downstream target genes of Wnt,such as c-myc and β-catenin.The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged.However,the enrichment of H4K20mel in the Wnt5a promoter and coding regions increased.In addition,genistein upregulated Phospho-cdc2,Mytl,Cyclin A,Cyclin E2,p21 and Phospho-histone H3,but downregulated Phospho-weel.Cell cycle arrest was induced in the G2/M phase.Conclusion:Genistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20mel in the Wnt5a gene promoter and coding regions,rather than demethylation.Genistein also blocks the cell cycle in the G2/M phase.Therefore,genistein is a potential anti-leukemia drug.

冷休克对PG、HeLa细胞周期调控及p21WAF1/cip1蛋白表达的影响

目的 研究冷休克对p5 3基因突变的PG细胞、p5 3野生型的HeLa细胞的周期调控及 p2 1WAF1/cip1蛋白表达的影响。方法 将PG、HeLa细胞置 4℃冷冻 4h ,使细胞发生冷休克 ,用流式细胞仪分析细胞DNA含量及凋亡细胞百分率 ,Westernblot检测 p2 1WAF/cip1蛋白的表达。 结果 冷休克诱导PG、HeLa细胞发生G2 /M期周期阻滞及凋亡 ,p2 1WAF/cip1蛋白表达增高 (PG以 12h、HeLa以 18h最为显著 )。冷休克诱导PG、HeLa细胞发生G2 /M期周期阻滞及凋亡与 p2 1WAF/cip1蛋白表达增高同步。结论 冷休克诱导PG、HeLa细胞发生G2 /M期阻滞及凋亡、p2 1WAF/cip1表达的增高与细胞的 p5 3状态无关。

电离辐射诱导的细胞G_2期阻滞

哺乳动物受X射线照射后,可以使细胞周期延迟或阻滞,包括G1期阻滞、S期延迟和G2期阻滞。G1期阻滞仅在野生型p53基因存在时出现,在清除DNA损伤的细胞中具有重要作用;而G2期阻滞更有利于损伤后DNA的修复和细胞存活,并且与p53基因存在状态无关。因此,对电离辐射诱导细胞G2期阻滞机制的探讨成为近年来国内外放射生物学领域的研究热点。

Discovery of Chrysoeriol,a PI3K-AKT-mTOR Pathway Inhibitor with Potent Antitumor Activity against Human Multiple Myeloma Cells in vitro

This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3,and its related molecular mechanisms.Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay.CCK-8 assay was employed to examine the growth inhibition rate and IC 50 (48 h) in peripheral blood mononuclear cells (PBMNCs),RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations.Cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE),and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software.The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC 50 concentration of chrysoeriol was adopted.The alterations in cell-cycle related proteins (Cyclin B1,Cyclin D1,p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis.The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol,resulting in cell cycle arrest in G 2 /M phase.Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein,meanwhile enhanced Cyclin B1 and p21 protein expression.Similar effects were not observed in PBMNCs from normal donors.It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor.It restrained the proliferation of human multiple myeloma cells,but didn’t affect proliferation of PBMNCs from normal donors.It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.

Ophiopogonin D inhibits cell proliferation,causes cell cycle arrest at G_2/M,and induces apoptosis in human breast carcinoma MCF-7 cells

OBJECTIVE： To investigate the effects of ophiopogonin D on human breast cancer MCF-7 cells METHODS： Cell viability was assessed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide and colony formation experiments. Cell cycle was measured with cell cycle flow cytometry and a living cell assay. Apoptosis and terminal deoxynucleoitidyl transferase-mediated dUTP nick end labeling assays were performed to detect the apoptosis of MCF-7 cells induced by ophiopogonin D. Finally, Western blotting was used to explore the mechanism. RESULTS： Exposure of cells to ophiopogonin D resulted in marked decreases in viable cells and colony formation with a dose-dependent manner. Treatment of these cells with ophiopogonin D also resulted in cell cycle arrest at the G2/M phase, and increased apoptosis. Mechanistically, ophiopogonin D-induced G2/M cell cycle arrest was associated with down-regulation of cyclin BI. Furthermore, activation of caspase-8 and caspase-9 was involved in ophiopogonin D-induced apoptosis. CONCLUSION： The data suggested that ophiopogonin D inhibits MCF-7 cell growth via the induction of cell cycle arrest at the G2/M phase.

HIV-1 Vpr protein activates the NF-κB pathway to promote G2/M cell cycle arrest

Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.

Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78-FOXM1-KIF20A pathway

Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.