Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri(L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.     

Simvastatin inhibits cell growth and induces apoptosis and G0/G1 cell cycle arrest in hepatic cancer cells

Hepatocellular carcinoma (HCC) is one of major health concerns worldwide and one of leading causes of cancer death after lung and gastric cancers. Simvastatin is a cholesterol-lowering drug which inhibits 3-hydroxy-3-methylglutarylcoenzyme CoA (HMG-CoA) reductase. Simvastatin exhibits numerous pleiotropic effects including anti-cancer activity. Yet, the anticancer effects in HCC remain poorly characterized. Therefore, in this study, we investigated the effects of simvastatin on tumor cell growth, apoptosis and cell cycle. HepG2 and Huh7 cell lines were treated with simvastatin (32 and 64 μM) for different time periods. Tumor cell growth was assessed using MTT assay. Apoptosis and cell cycle analysis were also evaluated. Analysis of cell cycle proteins involved in simvastatin-induced manipulation was performed by Western blot and quantitative RT-PCR analyses. Simvastatin induced a reduction of tumor cell growth. In both cell lines, simvastatin induced apoptosis and impaired cell cycle progression as depicted by the greater rates of G0/G1-phase cells than the rates of S-phase cells. Protein expression levels of cell cycle regulating proteins CDK1, CDK2, CDK4, cyclin D1, cyclin E, p19 and p27 were markedly altered by simvastatin. Moreover, CDC2, CCND1 and CDCN2D mRNA expressions were also altered by drug treatment. Collectively, these results suggest that simvastatin induces apoptosis in tumor cells and its anti-proliferative activity was accompanied by inhibition of cyclin-dependent kinases and cyclins, whereas CDK inhibitors p19 and p27 were enhanced. These results may provide novel insights into simvastatin tumor-suppressive action.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits the activation of antigen-specific T-cells in mice.

There are conflicting data in the literature regarding target cells in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity. In the present study, adult male C57BL/6 mice were exposed to TCDD (50 micrograms/kg) 4 days prior to immunization with ovalbumin (OVA). The effect of TCDD on the specific immune response in vivo was determined by T-cell proliferation and IL-2 production in response to either OVA or anti-mouse-CD3 antibodies plus PMA in vitro. The antigen-specific T-cell proliferation and IL-2 production in response to OVA were significantly suppressed by TCDD, while the polyclonal response to anti-CD3 antibodies plus PMA was not affected. This indicates that even at a high dose of TCDD the intra T-cell signalling pathways in resting cells are not disturbed, but TCDD selectively impairs the antigen-specific activation of T-cells. Since activated T-cells are required in antibody responses to T-dependent antigens, the low number of such cells observed in the present study, may well explain the suppressive effects of TCDD on humoral immunity reported previously.     

HCV NS2 protein inhibits cell proliferation and induces cell cycle arrest in the S-phase in mammalian cells through down-regulation of cyclin A expression

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2 protein is a HCV hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the functions of NS2 protein by examining its effects on cell growth and cell cycle progression. Stable NS2-expressing HeLa and Vero cell lines were established by transfection of the cells with pcDNA3.1(-)-NS2 followed by selection of the transfected cells in the presence of G418. We found that the proliferation rates of both NS2-expressing cell lines were inhibited by 40-50% compared with the control cells that were transfected with pcDNA3.1(-) control vector. Cell cycle analysis of these NS2-expressing cell lines shows that the proportion of cells in the S-phase increased significantly compared to that of control cells that do not express NS2 protein, suggesting NS2 protein induces cell cycle arrest in the S-phase. Further studies showed that the induction of cell cycle arrest in the S-phase by NS2 protein is associated with the decrease of cyclin A level. In contrast, the expression of NS2 protein does not affect the levels of cyclin-dependent kinase CDK2, CDK4, cyclin D1, or cyclin E. Our results suggest that HCV NS2 protein inhibits cell growth and induces the cell cycle arrest in the S-phase through down-regulation of cyclin A expression, which may be beneficial to HCV viral replication. Our findings not only provide information in the understanding mechanism of HCV infection, but also provide guidance for the future development of potential therapeutics for the prevention and treatment of the viral infection.     

Acute exposure of human lung cells to 1,3-butadiene diepoxide results in G1 and G2 cell cycle arrest

1,3-Butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2-treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2-treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 microM BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2-treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2-treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2-treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21(cip1) was observed in BDO2-treated cells, suggesting that the cell cycle arrest was p21(cip1)-mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects.     

G1/S cell cycle arrest provoked in human T cells by antibody to CD26

CD26 is T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity located in its extracellular region. The expression of CD26 is enhanced after activation of T cells, while it is preferentially expressed on a subset of CD4+ memory T cells in the resting state. In this paper, we demonstrate that binding of the soluble anti-CD26 monoclonal antibody (mAb) 1F7 inhibits human T-cell growth and proliferation in both CD26-transfected Jurkat T-cell lines and human T-cell clones by inducing G1/S arrest, which is associated with enhancement of p21Cip1 expression. This effect depends on the DPPIV enzyme activity of the CD26 molecule. Moreover, we show that expression of p21Cip1 after treatment with the anti-CD26 mAb 1F7 appears to be induced through activation of extracellular signal-regulated kinase (ERK) pathway. These data thus suggest that anti-CD26 treatment may have potential use in the clinical setting involving activated T cell dysregulation, including autoimmune disorders and graft-vs.-host disease.     

Erk1/2 promotes proliferation and inhibits neuronal differentiation of neural stem cells

Neural stem cells (NSCs) are in a complex niche in which cell-extrinsic cues and cell-intrinsic genetic mechanisms in chorus mediate their cellular processes such as self-renewal and differentiation. In this study, we found that inactivation of Erk1/2 with U0126 in NSCs significantly promoted neuronal differentiation and inhibited proliferation. Sustained Erk1/2 inactivity was required in this process. We also found that nerve growth factor (NGF) and collagen could promote the proliferation and inhibit neuronal differentiation by activating phosphorylation of Erk1/2. Cell-cycle regulators such as cyclin-dependent kinase 2 (Cdk2), Cyclin D1 and Hes1 mediated the effect of Erk on NSCs proliferation and differentiation. Our results showed that Erk1/2 played an important role in the interplay between cell-extrinsic cues and cell-intrinsic genetic mechanisms in neural stem cell biology.     

重组ACE2抑制人脐动脉平滑肌细胞前纤维蛋白-1表达及增殖

目的探讨重组血管紧张素转换酶2(ACE2)基因对血管平滑肌细胞前纤维蛋白-1(Profilin-1)表达及细胞增殖的影响。方法培养人脐动脉平滑肌细胞(HUASMC),用重组ACE2基因(rACE2)干预,进行血管紧张素Ⅱ(AngⅡ)刺激实验,分别用MTT法、实时定量PCR及Western blot检测HUASMC增殖与Profilin-1表达。结果与对照组相比,AngⅡ(100 nmol/L)刺激6 h后,HUASMC中Profilin-1表达明显增高(3.50±0.30 vs 1.00±0.10,P<0.05)。重组ACE2基因干预显著抑制上述增加(1.73±0.12 vs 3.50±0.30,P<0.05)。结论 ACE2基因过表达可明显逆转HUASMC中AngⅡ诱导的Profilin-1表达上调。     

人表皮生长因子受体显性负性突变体诱导胃癌细胞发生G0/G1期阻滞

 目的：探讨人表皮生长因子受体显性负性突变体（dominant negative epidermal growth factor receptor,DNEGFR）对胃癌细胞细胞周期的影响及其分子机制。方法：选用2株人胃癌细胞,分为如下6组：SGC-7901细胞未转染组（US组）、SGC-7901细胞pEGFP-N1质粒转染组(ES组)、SGC-7901细胞pEGFPN1-DNEGFR质粒转染组(DS组)、NCI-N87细胞未转染组（UN组)、NCI-N87细胞pEGFP-N1质粒转染组(EN组)和NCI-N87细胞pEGFPN1-DNEGFR质粒转染组(DN组)。采用流式细胞术检测细胞周期,Western blotting检测细胞周期素依赖性蛋白激酶2（CDK2）、cyclin D1、Ser9位点磷酸化糖原合成酶激酶3β［p-GSK-3β(Ser9)］、p21和p27蛋白水平。结果：转染pEGFPN1-DNEGFR质粒的人胃癌细胞株出现G0/G1期阻滞,CDK2、cyclin D1和p-GSK-3β（Ser9）蛋白水平降低,p21和p27蛋白水平则升高。结论：DNEGFR通过激活GSK-3β使cyclin D1蛋白水平降低,并降低CDK2蛋白水平,上调p21和p27蛋白水平,最终导致胃癌细胞发生G0/G1期阻滞。这一结果将为胃癌生物治疗研究提供新思路。     

miR-211通过靶向调控TFAM抑制人乳腺癌细胞系增殖

目的探讨miR-211靶向线粒体转录因子A(TFAM)对人乳腺癌细胞增殖的影响。方法用miR-211及TFAM作为研究对象。首先,在乳腺癌细胞中转染miR-211 mimics或miR-211抑制剂以实现miR-211过表达或miR-211沉默,并检测miR-211过表达或沉默时TFAM蛋白质的表达水平;其次,构建了在TFAM的5'端有或无6对碱基突变的荧光酶报告基因质粒(mut-TFAM/wt-TFAM),与miR-211 mimics或miR-211抑制剂共转染后检测荧光酶活性变化;然后,构建pc DNA3.1/TFAM质粒,与miR-211 mimics或miR-211抑制剂共转染后检测TFAM蛋白质表达水平变化;最后,检测pc DNA3.1/TFAM和mimics NC/miR-211 mimics共转染后乳腺癌细胞增殖的增殖。结果miR-211过表达抑制TFAM蛋白质表达(P0.01),miR-211沉默促进TFAM蛋白质表达(P0.01);miR-211可靶向结合TFAM调控其表达;pc DNA3.1/TFAM可实现TFAM过表达(mRNA P0.01,蛋白质P0.01),并可恢复miR-211对TFAM的抑制作用;miR-211可抑制乳腺癌细胞的增殖和增殖(P0.05),TFAM可促进乳腺癌细胞增殖增殖(P0.01),TFAM可回复miR-211对乳腺癌细胞增殖增殖的抑制作用(P0.05)。结论 miR-211靶向TFAM基因抑制人乳腺癌细胞的增殖。     

Akt抑制剂perifosine抑制胃癌细胞增殖并诱导凋亡的实验研究

目的: 探讨新的Akt抑制剂perifosine对胃癌细胞增殖与凋亡的影响。方法: 采用MTT法检测perifosine对胃癌细胞SGC-7901细胞增殖的影响;流式细胞术分析细胞周期变化;AnnexinⅤ-FITC试剂盒检测细胞凋亡;免疫印迹法检测蛋白表达水平。结果: Perifosine能够抑制SGC-7901细胞的增殖,且抑制作用呈时间和剂量依赖性。胃癌细胞经perifosine处理后,细胞阻滞于G₂期,p21表达水平增加,而cyclin B1的表达受到抑制;凋亡诱导现象随着perifosine剂量的增加而增强。免疫印迹结果显示perifosine活化SGC-7901细胞内caspase-3﹑caspase-9及其底物多聚(ADP-核糖)聚合酶(PARP),促进凋亡诱导蛋白Bax的表达,并抑制Bcl-2的表达。结论: Perifosine对人胃癌细胞SGC-7901的生长具有显著的抑制作用,其凋亡诱导活性与调节caspase家族和Bcl-2家族蛋白的表达密切相关。     

G1 arrest cell-cycle-dependent clastogenesis in UV-irradiated human fibroblasts

The demonstration of frequent allelic deletions in lung and colon cancers have reemphasized the importance of clastogenesis in carcinogenesis. We have investigated the mechanisms of induction of chromosome aberrations in ultraviolet-irradiated diploid human fibroblasts. Cells were irradiated with UV at various times during a parasynchronous wave of cell proliferation and then harvested during the first mitosis that followed irradiation. Metaphase spreads were stained with Geimsa and the yields of chromosome aberrations were quantified. Ultraviolet irradiation induced primarily chromatid-type chromosome aberrations which included chromatid breaks and exchanges. Frequencies of aberrations displayed significant differences according to the phase of the cell cycle in which irradiation occurred and the time after irradiation when metaphases were harvested. Fibroblasts that were irradiated when in G₀ and then immediately replated to stimulate cell division and cells that were at the S/G₂ border when irradiated displayed the fewest numbers of aberrations. For G₀-irradiated cells, the firts entering mitosis carried a higher frequency of aberrations than those collected 2–4 h later. In contrast, for S/G₂-irradaited cells the first into mitosis displayed fewere aberrations than subsequent fractions. Cells that were irradiated when at the G₁/S border displayed the greatest numbers of aberrations with the frequencies of chromatic exchanges being significantly increased over all other times of irradiation. These studies confirm that UV is an S-phase-dependent clastogen and point to the G₁/S border as a time of maximal sensitivity to clastogenesis. Irradiation of G₁ cells was shown to produce a fluence-dependent reduction in the rate of entry of cells into the S-phase. There appeared to be a point late in G₁ beyond which cells were resistant to irradiation and experienced less delay in S phase entry. Ataxia telangiectasia fibroblasts failed to delay entry to S phase following UV-irradiation in G₁ and displayed hypersensitivity to UV-induced chromosomal aberrations. The delay in entry of damaged cells into the S phase may have the beneficial effect of providing more time for repair of potentially clastogenic DNA damage before the onset of DNA replication.     

SP-TAT-Apoptin融合蛋白对HepG2细胞周期的影响

目的: 研究SP-TAT-Apoptin融合基因诱导人肝癌HepG2细胞凋亡和细胞周期阻滞, 探讨其用于肝癌治疗的可能性.方法: 通过DNA重组技术, 以pcDNA3.1/Apoptin质粒11为模板, 构建SP-TAT-Apoptin融合基因 plenti6-V5-D-TOPO真核表达载体.用SP-TAT-Apoptinplenti6-V5-D-TOPO真核表达载体转染中国仓鼠卵巢细胞(CHO), 转染后Blasticidin霉素进行筛选, 并进行鉴定得到稳定表达SP-TAT-Apoptin的CHO细胞株.收集含有TAT-Apoptin融合蛋白的筛选细胞培养上清, 用于HepG2细胞培养.于共培养后的不同时段收集HepG2细胞, 流式细胞术(FCM)检测细胞凋亡和细胞周期.结果: 用包含SP-TAT-Apoptin融合基因的plenti6-V5-D-TOPO真核表达载体瞬时转染中国仓鼠卵巢细胞(CHO), 成功筛选出稳定表达SP-TAT-Apoptin融合蛋白的CHO细胞, 收集细胞培养上清, 继续用于HepG2细胞培养, 可观察到HepG2细胞阻滞于G1期并引起细胞凋亡.结论: SP-TAT-Apoptin融合表达可引起HepG2细胞的细胞周期G1期阻滞.     

Knock-down of ABCE1 gene induces G1/S arrest in human oral cancer cells

Purpose: This study aims to explore the clinical characteristics of ATP binding cassette E1 (ABCE1) in oral squamous cell carcinomas (OSCC) and its roles in the proliferation, invasiveness, migration and apoptosis of the human oral squamous cell carcinoma cells CAL-27. Methods: The expression of ABCE1 and its target protein-RNase L, were first studied in tumor tissues of OSCC and adjacent non-tumor tissues. Moreover, CAL-27cells were transfected by ABCE1-specific shRNA, then MTT assay, the transwell and scratch assay were used to study cell proliferation and migration activity; the apoptosis rate and cell cycle distribution were tested by flow cytometry. Western blot and RT-PCR assay were adopted to measure their silencing efficacy. Results: ABCE1 expression is low in the adjacent non-tumor tissues while the expression is high in the oral cancer; the expression is reversely proportional to the differentiation degrees. The expression of RNaseL was in contrary to ABCE1. After transfected with ABCE1-siRNA, the proliferation, invasiveness and migration capabilities of cells decreased significantly whilst the apoptosis rate enhanced greatly (P < 0.01). Meanwhile, the expression of ABCE1 in CAL-27 cells was blocked (P < 0.01) while the expression of RNase L increased significantly (P < 0.01). Conclusion: ABCE1 is closely connected with the pathogenesis and development of oral cancer, which acts through the cellular pathways of 2-5A/RNase L.     

脱落酸抑制人肝癌细胞增殖

目的探讨脱落酸抑制SMMC-7721人肝癌细胞增殖作用的机制。方法免疫细胞化学检测P53、Ki-67、Cyclin D1蛋白表达;RT-PCR检测P53和端粒酶mRNA表达。结果经脱落酸(ABA)、环六亚甲基二乙酰胺(HMBA)和ABA HMBA作用后细胞内mtP53、Cyclin D1、Ki-67及细胞内mt P53、hTERT mRNA表达水平均明显降低(P<0.05)。结论ABA抑制SMMC-7721人肝癌细胞增殖,其作用机制可能是调控细胞周期,降低mtP53、Ki-67、Cyclin D1蛋白及mtP53,hTERT mRNA表达。