A tumor cell growth inhibitor from Saposhnikovae divaricata

In the present study, we tested ethanolic extracts from 10 Chinese herbs for their effects on K562, Raji, Wish, HeLa, Calu-1, and Vero tumor cells proliferation. On a percentage basis, panaxynol purified from Saposhnikovae divaricata had the highest inhibitory activity on various tumor cells proliferation. Cell-cycle analysis indicated that panaxynol arrested the cell cycle progression of tumor cells from the G1 transition to the S phase. In an attempt to further localize the point in the cell cycle where arrest occurred, gene expression of cyclin E, a key regulatory event leading to the G1/S boundary was examined. Results indicated that the levels of cyclin E mRNA in various tumor cells were decreased by panaxynol. Thus, the suppressant effects of panaxynol on proliferation of various tumor cells appeared to be mediated, at least in part, through impairments of cyclin E mRNA levels and arresting cell cycle progression in the cells.     

shRNA靶向沉默Eag 1钾通道对骨肉瘤细胞增殖、细胞周期及cyclin D1／E表达的影响

目的 探讨短发夹RNA（shRNA）靶向沉默Ether-go-go 1（Eag1）钾通道对骨肉瘤MG-63细胞增殖、细胞周期及cyclin D1／E表达的影响。 方法 使用成功构建的Ad5-Eag1-shRNA表达载体转染MG-63细胞（抑制组）,同时设转染无义序列（Ad5-Control-shRNA）的空转染组及不进行任何处理的对照组。采用逆转录聚合酶链式反应和Western blotting检测Ad5-Eag1-shRNA转染骨肉瘤MG-63细胞后的Eag1 mRNA和蛋白水平,分别采用CCK-8法和克隆形成实验检测各组的细胞增殖情况,流式细胞仪和Western blotting分别检测各组的细胞周期变化和细胞周期蛋白（cyclin D1和cyclin E）的蛋白水平。建立裸鼠骨肉瘤异种移植模型并测量各组裸鼠肿瘤体积的变化情况。结果 抑制组的Eag1 mRNA和蛋白水平均低于空转染组和对照组,差异有统计学意义（P＜0.05）;与其余两组相比,抑制组的细胞增殖、克隆形成数、S期细胞比例及细胞周期蛋白水平均降低,而G0／G1期细胞比例升高,以上差异均有统计学意义（P＜0.05）。抑制组裸鼠的瘤体积小于空转染组和对照组（P＜0.05）。结论 通过shRNA抑制Eag1基因表达可抑制MG-63细胞的增殖并诱导G0／G1期阻滞,可能与调控cyclin D1／E通路有关,有望成为骨肉瘤治疗和诊断的新靶点。     

Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.     

稳定表达Cyclin E细胞系的建立及Cyclin E高表达对人乳腺癌MCF 7细胞生长及细胞周期的影响

 目的 观察cyclin E 的高表达对乳腺癌细胞MCF 7 生长及周期的影响。方法 构建cyclin EcDNA真核表达载体并采用lipofectAMINE 转染方法将其导入MCF 7 细胞,获得稳定表达cyclin E的细胞系。通过对细胞生长曲线绘制、3H TdR测定及细胞周期分布等的分析,观察其对细胞生长、增殖的影响。结果 cyclin E的高表达可以使细胞的生长速度加快（ 约为对照1-52 倍） 及DNA 掺入增加（4-2 倍）,G1 S移行加速;pRB的磷酸化形式的增多（3 倍）.结论 cyclin E的高表达可明显影响MCF 7 细胞的生长、增殖,并可能通过pRB 磷酸化,影响细胞周期G1 S期移行而实现其对生长的调节。     

E2F3a在U251胶质瘤细胞中的功能研究

背景与目的：E2F3a是一种重要的转录激活因子，在多种肿瘤组织中均呈现表达上调。E2F3a在恶性化程度较高的神经胶质瘤中表达较高，目前它在胶质瘤细胞中的功能尚不清楚。本文的目的是研究E2F3。对U251胶质瘤细胞周期和凋亡的影响。方法：通过脂质体介导质粒转染在U251细胞中过表达E2F3a：用Western blotting检测细胞内E2F3a蛋白质的表达水平：用PT-核DNA染色结合流式细胞术分析细胞周期分布：用Annexin V—PE和7-AAD染色结合流式细胞术分析细胞凋亡比例；用实时荧光定量PCR方法测定细胞内A2、B1、D1和E细胞周期素mRNA的相对表达水平。结果：与空载体转染对照组相比．E2F3a过表达可将位于S期的细胞比例提高28％（P〈0．01），将位于G0/G1期和G2/M的细胞比例分别降低15％和13％（P〈0．01）。E2F3a过表达对细胞凋亡无明显影响。进一步研究发现，E2F3a可显著上调细胞周期素D1和E的mRNA表达．而不影响周期素A2和B1的mRNA表达。结论：E2F3a是胶质瘤细胞增殖的促进因子，它可通过上调细胞周期素D1和E的表达加速细胞由G，期进入S期。     

Garcinol,an Acetyltransferase Inhibitor,Suppresses Proliferation of Breast Cancer Cell Line MCF-7 Promoted by 17β-Estradiol

The acetyltransferase inhibitor garcinol, a polyisoprenylated benzophenone, is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Anti-cancer activity has been suggested but there is no report on its action via inhibiting acetylation against cell proliferation, cell cycle progression, and apoptosis-inhibtion induced by estradiol (E2) in human breast cancer MCF-7 cells. The main purposes of this study were to investigate the effects of the acetyltransferase inhibitor garcinol on cell proliferation, cell cycle progression and apoptosis inhibition in human breast cancer MCF-7 cells treated with estrogen, and to explore the significance of changes in acetylation levels in this process. We used a variety of techniques such as CCK-8 analysis of cell proliferation, FCM analysis of cell cycling and apoptosis, immunofluorescence analysis of NF-κB/p65 localization, and RT-PCR and Western blotting analysis of ac-H3, ac-H4, ac-p65, cyclin D1, Bcl-2 and Bclxl. We found that on treatment with garcinol in MCF-7 cells, E2-induced proliferation was inhibited, cell cycle progression was arrested at G0/G1 phase, and the cell apoptosis rate was increased. Expression of ac-H3, ac-H4 and NF-κB/ac-p65 proteins in E2-treated MCF-7 cells was increased, this being inhibited by garcinol but not ac-H4.The nuclear translocation of NF-κB/p65 in E2-treated MCF-7 cells was also inhibited, along with cyclin D1,Bcl-2 and Bcl-xl in mRNA and protein expression levels. These results suggest that the effect of E2 on promoting proliferation and inhibiting apoptosis is linked to hyperacetylation levels of histones and nonhistone NF-κB/p65 in MCF-7 cells. The acetyltransferase inhibitor garcinol plays an inhibitive role in MCF-7 cell proliferation promoted by E2. Mechanisms are probably associated with decreasing ac-p65 protein expression level in the NF-κB pathway, thus down-regulating the expression of cyclin D1, Bcl-2 and Bcl-xl.     

Insufficient effect of p27(KIP1) to inhibit cyclin D1 in human esophageal cancer in vitro

The cell cycle is controlled by protein complexes composed of cyclins and cyclin-dependent kinases. p27KIP1 (p27) is one of the Kip/Cip family cyclin-dependent kinase inhibitory proteins which negatively regulate cell cycle progression, and have been proposed as candidate tumor suppressor genes. To examine the role of p27 in the development of human esophageal squamous cell carcinoma (ESCC), we performed Western blot and immunoprecipitation analyses of the levels of expression of p27 protein in a series of ESCC cell lines. This protein was expressed at various levels in these cell lines during exponential growth. p27 level was significantly associated with that of cyclin D1, but not of cyclin E. Further cell cycle synchronization studies demonstrated that p27 was free or bound with affinity to cyclin E-CDK2 more than to cyclin D1-CDK4 or cyclin D1-CDK6. It is known that overexpression of cyclin D1 rather than cyclin E is involved in the pathogenesis of ESCC. Our findings indicated that high expression of p27 throughout the G1 to S phase may inhibit more likely cyclin E, than cyclin D1, which promotes tumor growth of esophageal squamous cell carcinoma.     

Cancerous inhibitor of protein phosphatase 2A contributes to human papillomavirus oncoprotein E7-induced cell proliferation via E2F1

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in many human malignant tumors including cervical cancer. Human papillomavirus (HPV) oncoprotein E7 is the key transformation factor in cervical cancer. Our previous data showed a positive association of CIP2A and HPV-16E7 protein levels; however, how CIP2A is regulated by HPV-E7 and the roles of CIP2A in HPV-E7-mediated cell proliferation are unknown. In this study, we demonstrated that HPV-16E7 protein significantly upregulating CIP2A mRNA and protein expression depended on retinoblastoma protein pRb rather than p130. CIP2A siRNA knockdown in HPV-E7-expressing cells inhibited cell proliferation, DNA synthesis and G1/S cell cycle progression. CIP2A siRNA decreased the protein levels of cyclin-dependent kinase 1 (Cdk1), Cdk2 and their partner cyclin A2, with no change in levels of Cdk4, Cdk6 and their partner cyclin D1. The downregulation of Cdk1 and Cdk2 was independent of c-Myc; instead, E2F1 was the main target of CIP2A in this process, as overexpression of E2F1 rescued the inhibitory effects of CIP2A siRNA knockdown on cell proliferation and G1 arrest of HPV-E7-expressing cells. Our studies reveal a novel function of CIP2A in HPV-16E7-mediated cell proliferation.     

PSAT1 regulates cyclin D1 degradation and sustains proliferation of non‐small cell lung cancer cells

Multiple nodes in the one‐carbon metabolism pathway play important regulatory roles in cancer cell growth and tumorigenesis. The specific biological functions of metabolic enzymes in regulating the signaling pathways that are associated with tumor cell growth and survival, however, remain unclear. Our current study found that phosphoserine aminotransferase 1 (PSAT1), an enzyme catalyzing serine biosynthesis, was significantly up‐regulated in non‐small cell lung cancer (NSCLC) and was involved in the regulation of E2F activity. Loss‐ and gain‐of‐function experiments demonstrated that PSAT1 promoted cell cycle progression, cell proliferation and tumorigenesis. Mechanistic study suggested that elevated PSAT1 led to inhibition of cyclin D1 degradation and subsequently an alteration in Rb‐E2F pathway activity, which in turn enhanced G1 progression and proliferation of NSCLC cells. Moreover, phosphorylation of cyclin D1 at threonine 286 by GSK‐3β was required for PSAT1‐induced blockage of cyclin D1 degradation. We also found that the activity of p70S6K mediated the effects of PSAT1 on GSK‐3β phosphorylation and cyclin D1 degradation. We further identified that PSAT1 was over‐expressed in NSCLC and predicted poor clinical outcome of patients with the disease. Correlation analysis showed that PSAT1 expression positively correlated with the levels of phosphorylated GSK‐3β, cyclin D1 and phosphorylated Rb in NSCLC primary tumors. These findings uncover a mechanism for constitutive activation of E2F via which unrestrained cell cycle progression occurs in NSCLC and may represent a prognostic biomarker and therapeutic target.     

Possible mechanism of growth inhibition by Scutellaria baicalensis in an estrogen-responsive mouse tumor cell line

We have studied the effects of Saiboku-to, a traditional Chinese medicine having suppressive activities for leukotriene production and release, on the proliferation of the estrogen-responsive mouse Leydig tumor cell line B-1F. In our previous reports, it is shown that Saiboku-to promotes, but Scutellaria baicalensis, one of the components (herbs) of Saiboku-to, significantly inhibits the proliferation of B-1F cells in?vitro and in?vivo, and induces DNA fragmentation and morphological changes such as nuclear aggregation and fragmentation. In this study, we examined telomerase activity, cell cycle, polyunsaturated fatty acid metabolism and expression of nuclear factor κB (NF-κB) in order to determine the mechanism of growth inhibition in B-1F cells treated with Scutellaria baicalensis. Telomerase activity was decreased in a dose-dependent manner in treated B-1F cells. Cellular populations in the sub-G0/G1 and G2/M phases were increased, but those in M phase had no change. Although cyclin D1 mRNA was highly expressed in the presence of estradiol (E2), cyclin A and E mRNA levels did not significantly change. When B-1F cells were treated with Scutellaria baicalensis, expression of cyclin D1 was suppressed and that of p21 was inversely increased. Moreover, Scutellaria baicalensis influenced arachidonic and linoleic acid metabolism, and increased production of 13(S)-HODE. In the presence of E2 Scutellaria baicalensis decreased expression of NF-κB p65 to 0.71-fold in B-1F cells. These results show that Scutellaria baicalensis might induce cell cycle arrest at G1 phase and apoptosis via inhibition of telomerase activity, changes of enzymatic activities in polyunsaturated fatty acid metabolism and suppression of NF-κB.     

Intranuclear compartmentalization of cyclin E during the cell cycle: disruption of the nucleoplasm-nucleolar shuttling of cyclin E in bladder cancer

Cyclin E/cyclin-dependent kinase 2 complexes are essential during the cell cycle for entrance into S phase. Cyclin E expression starts in mid-G1, reaches a maximum at S-phase entrance, and undergoes proteolysis mediated by the ubiquitin pathway as the cell progresses through S phase. Laser scanning cytometry, a microscope-based cytofluorometer combining the advantages of both flow and image analysis, allowed the determination of subcellular localization of cyclin E, p27, and retinoblastoma protein during cell cycle progression in normal human fibroblasts and nine bladder cancer cell lines. We observed that in normal fibroblasts and most tumor cell lines, cyclin E localizes in the nucleoplasm during mid-G1, and is translocated to the nucleolus during G1-S-phase transition, and its levels are undetectable in G2-M phase. Neither levels nor subcellular localization of p27 and retinoblastoma protein was cell cycle dependent in normal or tumor cells. However, four of nine bladder cancer cell lines continued to express cyclin E in all phases of the cycle, and image analysis revealed that it was localized to nucleoli. These observations suggest that the nucleolus mediates a cyclin E "shuttling" between the nucleus and the cytoplasm that is probably involved in its regulation and that this mechanism could be disrupted in bladder cancer.     

Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte

We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC50, approximately 30 microM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G1 phase in HL-60 human promyelocytic leukemia cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21(CIP1/WAF1) and p27(KIP1), were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21(CIP1/WAF1) and p27(KIP1) to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G1 arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21(CIP1/WAF1) and p27(KIP1) levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.     

RNA interference targeting of A1 receptor-overexpressing breast carcinoma cells leads to diminished rates of cell proliferation and induction of apoptosis

To determine if A1 adenosine receptors mediate breast tumorigenesis, we evaluated A1 receptor expression in human tumor cell lines and human primary breast tumor tissues using both quantitative RT-PCR and Western blot analysis. A1 receptor mRNA expression is upregulated in all breast tumor cell lines examined (n=7) compared to normal mammary epithelial cells/cell lines (n=3) as determined by quantitative RT-PCR analysis. Western blot analysis indicates that protein expression of A1 adenosine receptor is higher in 15 (62.5%) of 24 human primary breast tumor tissues than in matched normal breast tissue. To explore its cellular function, the A1 adenosine receptor was depleted by small interfering RNA (siRNA) in MDA-MB-468 human breast tumor cells. Depletion of A1 receptors in MDA-MB-468 breast tumor cells attenuated both cell growth and cell proliferation as measured by cell number counts and [(14)C]-thymidine incorporation, respectively. Cell cycle analysis indicated that depletion of A1 receptors by siRNA impairs G(1) checkpoint, leading to marked accumulation of cells in G(2)/M phase, in agreement with the inhibitory effect on cell proliferation. Further supporting this finding, synchronization studies of Hela cells in various cell cycle phases suggest that A1 receptor expression is suppressed in G(2)/M cells and depletion of A1 receptor expression by siRNA produced differential expression of several key cell cycle regulators, i.e., accumulation of the cyclin-dependent kinase inhibitor p27 with concomitant reduction of CDK4 and cyclin E proteins. In addition to the impact on cell cycle progression, depletion of A1 receptors by siRNA results in substantial cell death and apoptosis as determined by FACS analysis and annexin V staining method. Together these findings suggest that the A1 adenosine receptor may contribute to tumor cell growth and survival in breast tumor cells.     

干扰B7-H4表达通过下调E2F家族相关转录因子抑制乳腺癌细胞的增殖

目的：探讨干扰B7-H4表达对乳腺癌细胞增殖、凋亡、周期以及相关下游分子表达的影响。方法：利用脂质体转染技术分别将特异性靶向B7-H4的siRNA(siB7-H4)及其阴性对照（siNC）转染至对数生长期的乳腺癌T47D和MCF-7细胞,分别命名为T47D-siB7-H4、T47D-siNC、MCF-7-siB7-H4和MCF-7-siNC组。用qPCR法和WB法验证siRNA干扰效果及其对细胞周期分子cyclin D1表达的影响,CCK-8法和FCM分别检测干扰B7-H4表达对T47D和MCF-7细胞增殖、周期和凋亡的影响,qPCR法检测B7-H4干扰对E2F家族相关转录因子表达的影响。结果：成功构建干扰B7-H4表达的乳腺癌T47D和MCF-7细胞。与T47D-siNC和MCF-7-siNC组相比,T47D-siB7-H4和MCF-7-siB7-H4组细胞中B7-H4 mRNA和蛋白表达水平均显著降低、细胞增殖能力显著降低（均P<0.01）,并伴有G1/S期细胞周期阻滞以及cyclin D1表达下调（均P<0.01）,但细胞凋亡率差异无统计学意义（均P>0.05）...