Imperatorin sensitizes anoikis and inhibits anchorage-independent growth of lung cancer cells

The anoikis-sensitization activity of imperatorin, an active furanocoumarin component of Angelica dahurica root, is reported herein for the first time. The present study demonstrated that the imperatorin treatment at sub-toxic concentrations enhanced human lung cancer H23 cell apoptosis after detachment. A Western blot analysis showed that imperatorin significantly enhanced the p53 protein level, which subsequently down-regulated Mcl-1 protein and up-regulated Bax, while it had a minimal effect on Bcl-2 expression. In addition, an anchorage-independent growth assay was performed to support the anti-metastasis potential of imperatorin. Consistent with anoikis assay, imperatorin exhibited a strong inhibitory effect on the anchorage-independent growth of the cells. Further, this study demonstrated that imperatorin sensitizes anoikis in other lung cancer cells, namely, H292 and A549. Because anoikis was shown to be a critical hindrance in preventing cancer cell metastasis, the knowledge regarding such an activity and an underlying mechanism may lead to the development of this compound for a cancer therapy.     

Pentoxifylline inhibits platelet-derived growth factor-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation

Pentoxifylline (PTX) is a potent inhibitor of mesangial cell proliferation, but its underlying mechanism is poorly understood. Here, we demonstrate that in platelet-derived growth factor (PDGF)-stimulated mesangial cells, PTX causes G1 arrest by down-regulation of cyclin D1 expression, which subsequently attenuates Cdk4 activity. In vivo, PTX similarly reduces cyclin D1 expression in mesangial cells of rats with acute Thy1 glomerulonephritis. The mechanism by which PTX reduces cyclin D1 is also investigated. PTX blocks Akt but not phosphatidylinositol 3-kinase (PI3K) activation in response to PDGF and abrogates cyclin D1 induction by PI3K, suggesting an effect of PTX on Akt itself. Indeed, PTX is capable of blocking the membrane translocation of Akt, and enforced targeting of Akt to cell membrane prevents the inhibition of Akt and cyclin D1 by PTX. Because PTX is known to increase intracellular cAMP levels by inhibiting phosphodiesterase, the role of protein kinase A (PKA) in these events is investigated. The PKA antagonist N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) abolishes cell proliferation effects of PTX and restores cyclin D1 expression as well as Akt membrane translocation and activation by PDGF, whereas dibutyryl cAMP and forskolin recapitulate the functions of PTX in mesangial cells. In conclusion, our results indicate that PTX, acting through PKA, interferes with PDGF signaling to Akt activation by blocking Akt membrane translocation, thereby inhibiting cyclin D1 expression and mesangial cell proliferation.     

细胞周期素E、D1与增殖细胞核抗原在肝癌中的表达及临床意义

目的研究肝癌组织中细胞周期素E（cyclinE）、细胞周期素D1（cyclinD1）与增殖细胞核抗原（PCNA）的表达情况,探讨其在肝癌发生发展中的作用及临床意义。方法用免疫组化链霉菌素一生物素（SP）法,对术前未使用过化疗、放疗及免疫治疗的60例肝癌组织及35例癌旁肝组织进行检测。结果cyclinE、cyclinD1与PCNA的表达在肝癌组织中高于癌旁肝组织（P〈0．01）,在分化差的和有转移的肝癌中表达高于分化好的和元转移的,而且cyclinE与cyclinD1的表达密切相关（P〈O．01）。结论cyclinE、cyclinD1与PCNA表达与肝癌的增殖、分化、转移有关,其表达状况可以作为判断恶性程度及预后的指标。     

Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells

Betulinic acid (BA) is a pentacyclic triterpene found in many plant species, among others in the bark of white birch Betula alba. BA was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial and anti-inflammatory activities, and in particular to inhibit growth of cancer cells. The aim of the study was further in vitro characterization of BA anticancer activity. In this study, we demonstrated a remarkable antiproliferative effect of BA in all tested tumor cell cultures including neuroblastoma, rabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukemia and multiple myeloma, as well as in primary cultures isolated from ovarian carcinoma, cervical carcinoma and glioblastoma multiforme. Furthermore, we have shown that BA decreased cancer cell motility and induced apoptotic cell death. We also observed decrease of bcl2 and cyclin D1 genes expression, and increase of bax gene expression after betulinic acid treatment. These findings demonstrate the anticancer potential of betulinic acid and suggest that it may be taken into account as a supportive agent in the treatment of cancers with different tissue origin.     

FLT3靶向RNA干扰对HL-60细胞增殖凋亡及cyclinD1、cyclinA表达的影响

目的探讨FLT3靶向RNA干扰对HL-60细胞增殖、凋亡及cyclinD1、cyclinA表达的影响。方法体外构建FLT3靶向短发夹状干扰RNA(FLT3-shRNA),转染HL-60细胞,RT-PCR、流式细胞术(FCM)鉴定FLT3的表达;CCK-8法检测细胞增殖活力;FCM检测细胞周期;AnnexinV染色法检测凋亡;RT-PCR及Western blot检测cyclinD1,cyclinA在mRNA、蛋白水平的表达。结果FLT3-shRNA转染可下调FLT3的表达,它对FLT3 mRNA和蛋白的抑制率分别达(81.66±10.25)%,(76.76±11.23)%。FLT3表达下降后细胞增殖活力受到抑制,转染48h抑制率达(31.66±2.97)%,72h达(33.10±3.43)%;细胞生长曲线低平,缺乏指数增殖特征。与对照组比,转染48h细胞周期重新分布,G0/G1期(58.48±6.17)%的明显上升,S期(27.72±5.10)%下降;细胞早期凋亡率(8.95±0.88)%上升;细胞内cyclinD1的mRNA、蛋白表达下降,cyclinA的表达无明显改变。结论FLT3靶向RNA干扰通过下调cyclinD1表达有效地抑制细胞增殖,具有潜在的临床应用价值。     

Anicemycin, a new inhibitor of anchorage-independent growth of tumor cells from Streptomyces sp. TP-A0648

The anchorage-independence of cells is closely related to their tumorigenicity. In the screening of inhibitors of anchorage-independent growth of tumor cells, anicemycin was isolated from the fermentation broth of an actinomycete strain TP-A0648. The producing strain was isolated from a leaf of Aucuba japonica collected in Toyama, Japan and identified as Streptomyces sp. based on the taxonomic data. The structure of anicemycin was elucidated as a new analog of spicamycin by NMR and MS analysis. Anicemycin inhibited the anchorage-independent growth of the human ovary cancer SKOV-3 cells with an IC50 of 0.015 microM about three times more potently than their anchorage-dependent growth.     

Anicequol,a novel inhibitor for anchorage-independent growth of tumor cells from Penicillium aurantiogriseum Dierckx TP-F0213

A novel inhibitor for anchorage-independent growth of tumor cells was isolated from the culture broth of a fungal strain. The producing strain TP-F0213 was identified as Penicillium aurantiogriseum Dierckx based on the taxonomic study. The compound designated anicequol was obtained by solvent extraction, HP-20 and silica gel chromatographies and recrystallization. The planar structure was elucidated by NMR analysis to be 16-acetoxy-3,7,11-trihydroxyergost-22-en-6-one. The absolute configuration was determined by the X-ray analysis of 3,7-bis-p-bromobenzoyl derivative. The carbon skeleton of anicequol has the same absolute configuration as ergostane and the configurations of substituents are 3beta, 5alpha, 7beta, 11beta, 16beta and 24S. Anicequol inhibited the anchorage-independent growth of human colon cancer DLD-1 cells with the IC50 of 1.2 microM whereas the IC50 against anchorage-dependent growth was 40 microM.     

Let-7a靶向调节周期素D2抑制胶质瘤细胞增殖

目的探讨let-7a通过靶向调控周期素D2(cyclin D2)对胶质瘤细胞增殖的影响。方法在U87和U251胶质瘤细胞中转染入化学合成的let-7a模拟物上调let-7a表达,荧光素酶报告基因实验检测let-7a与周期素D2的靶向调控关系,Western blot检测转染let-7a后周期素D2的表达情况,MTT和流式细胞术检测let-7a对胶质瘤细胞增殖的抑制作用。结果在胶质瘤细胞中周期素D2是let-7a的靶基因,过表达的let-7a可下调周期素D2表达和抑制胶质瘤细胞增殖。结论let-7a可通过靶向调控周期素D2而抑制胶质瘤细胞增殖。     

Let-7a inhibits proliferation of glioma by target regulation of cyclin D2

目的 探讨let-7a通过靶向调控周期素D2 (cyclin D2)对胶质瘤细胞增殖的影响.方法 在U87和U251胶质瘤细胞中转染入化学合成的let-7a模拟物上调let-7a表达,荧光素酶报告基因实验检测let-7a与周期素D2的靶向调控关系,Western blot检测转染let-7a后周期素D2的表达情况,MTT和流式细胞术检测let-7a对胶质瘤细胞增殖的抑制作用.结果 在胶质瘤细胞中周期素D2是let-7a的靶基因,过表达的let-7a可下调周期素D2表达和抑制胶质瘤细胞增殖.结论 let-7a可通过靶向调控周期素D2而抑制胶质瘤细胞增殖.     

Inhibition of the cyclin D1/E2F pathway by PCA-4230, a potent repressor of cellular proliferation

1. Tight control of cellular growth is essential to ensure normal tissue patterning and prevent pathological responses. Excessive vascular smooth muscle cell (VSMC) proliferation is associated with the pathophysiology of atherosclerosis and restenosis post-angioplasty. Thus, drug targeting of pathological VSMC growth may be a suitable therapeutic intervention in vascular proliferative diseases. 2. In the present study, we investigated the mechanisms underlying VSMC growth arrest induced by the pharmacological agent PCA-4230. Addition of PCA-4230 to cultured VSMCs blocked the induction of cyclin D1 and cyclin A expression normally seen in serum-restimulated cells. Moreover, PCA-4230 inhibited cyclin-dependent kinase 2 (CDK2) activity and abrogated hyperphosphorylation of the retinoblastoma (Rb) gene product. Similarly, PCA-4230-dependent growth arrest of transformed cell lines correlated with reduced level of cyclin D1 protein and inhibition of CDK2 activity. Consistent with these findings, PCA-4230 repressed serum-inducible cyclin A promoter activity, and overexpression of either cyclin D1 or E2F1 efficiently circumvented this inhibitory effect. Importantly, adenovirus-mediated overexpression of E2F1 restored S-phase entry in PCA-4230-treated VSMCs, demonstrating that PCA-4230 represses cyclin A gene expression and VSMC growth via inhibition of the cyclin D1/E2F pathway. 3. Because of its ability to inhibit the growth of human VSMCs and transformed cell lines, future studies are warranted to assess whether PCA-4230 may be a suitable therapeutic intervention for the treatment of hyperproliferative disorders, including cardiovascular disease and cancer.     

生长抑素类似物抑制胆管癌细胞增殖的研究

目的探讨生长抑素类似物奥曲肽(OCT)对人胆管癌细胞细胞株QBC939的抑制作用及可能的作用机制。方法四氮唑蓝(MTT)法检测OCT对人胆管癌细胞株QBC939增殖的影响,流式细胞仪检测OCT对细胞周期变化的作用,免疫组织化学法研究OCT对人胆管癌细胞株QBC939P27KIP1蛋白表达变化的影响。建立胆管癌裸鼠模型,进一步探讨生长抑素的作用。结果MTT法检测不同浓度OCT(5、0.5、0.05、0.005mg/L)作用于QBC93948h后,实验组0.5、5mg/L组和对照组吸光度(A)值比较差异有统计学意义(P<0.05)。细胞生长率分别为83.80%,80.28%,77.53%,65.32%。流式细胞仪分析显示不同浓度OCT作用于QBC93948h后,G0/G1期细胞明显增多(P<0.05)。免疫组织化学P27KIP1蛋白表达随OCT浓度的增加而增强(P<0.05)。建立胆管癌裸鼠模型后,OCT干预21d后实验组瘤重明显小于对照组(P<0.05)。结论OCT可抑制人胆管癌细胞的增殖。在一定范围内呈剂量依赖性,其抗肿瘤作用机制主要是通过细胞周期阻滞来实现的,而这一作用的实现可能与P27KIP1蛋白表达的上调有关。     

Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1

Background and purpose:

Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation.

Experimental approach:

Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng·mL⁻¹) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators.

Key results:

Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC₅₀= 520 ± 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 ± 8%) and asthmatic subjects (57 ± 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21^Waf/Cip-1, p27^Kip1, cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC₅₀= 450 ± 100 pM) PDGF-induced cell cycle progression.

Conclusions and implications:

Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.This Research Paper is the subject of a Commentary in this issue by Clifford and Knox (pp. 1426–1428). To view this article visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Huanglian, A chinese herbal extract, inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer cells

Huanglian is an herb that is widely used in China for the treatment of gastroenteritis. We elected to determine whether huanglian could inhibit tumor cell growth by modulating molecular events directly associated with the cell cycle. Huanglian inhibited tumor growth and colony formation of gastric, colon, and breast cancer cell lines in a time- and dose-dependent manner. Cell growth was completely inhibited after 3 days of continuous drug exposure to 10 microg/ml of herb. This degree of growth inhibition was significantly greater than that observed with berberine, the major constituent of the herb. The inhibition of cell growth by huanglian was associated with up to 8-fold suppression of cyclin B1 protein. This resulted in complete inhibition of cdc2 kinase activity and accumulation of cells in G(2). The mRNA expression of cyclin B1 was not changed after huanglian treatment. There was no change in the protein expression of cyclins A or E. Therefore, the effect of huanglian on inhibiting tumor growth seems to be mediated by the selective suppression of cyclin B1, which results in the inhibition of cdc2 kinase activity. Inhibition of cyclin dependent kinase (cdk) activity is emerging as an attractive target for cancer chemotherapy. Huanglian represents a class of agents that can inhibit tumor cell growth by directly suppressing the expression of a cyclin subunit that is critical for cell cycle progression. These results indicate that traditional Chinese herbs may represent a new source of agents designed for selective inhibition of cyclin dependent kinases in cancer therapy.     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.     

ErbB-2-induced mammary tumor growth: the role of cyclin D1 and p27Kip1

The neu (c-erbB-2, HER2) proto-oncogene encodes a receptor tyrosine kinase that is a member of an important growth factor receptor family which includes the epidermal growth factor receptor (EGFR, ErbB1), ErbB3 and ErbB4. The neu is found over-expressed in 20-30% of human breast tumors. The c-erbB-2 is sufficient for the induction of mammary tumorigenesis in transgenic mice and the pathology of these mammary tumors strongly resembles human breast cancer. Murine transgenic models engineered to recapitulate human breast cancer provide an excellent and straightforward approach to dissect the molecular mechanisms governing the onset and progression of this disease. The molecular mechanisms by which ErbB-2 transforms cells involves direct effects on components of the cell-cycle regulatory apparatus. Recent studies have demonstrated a key role for components of the cell-cycle, in particular cyclin D1 and p27Kip1 (p27) in the onset and progression of ErbB-2-induced murine mammary tumorigenesis. Such studies have provided further impetus to therapeutics targeting these cell-cycle proteins.     

星形细胞胶质瘤中生存素、周期蛋白E和端粒酶相关蛋白的表达与意义

目的 探讨生存素(survivin)、周期蛋白E(cyclin E)和端粒酶相关蛋白(hTP1)在星形细胞胶质瘤中的表达与意义.方法 应用免疫组织化学SP法、原位杂交法分别检测106例星形细胞胶质瘤组织中生存素、周期蛋白E和hTP1的表达, 分析它们之间的相互关系.结果 生存素定位于肿瘤细胞胞浆,周期蛋白E和hTP1主要定位于肿瘤细胞核.生存素、周期蛋白E 和hTP1的阳性表达率分别为55.7%(59/106)、61.3%(65/106)和84.9%(90/106).生存素、周期蛋白E表达和hTP1标记指数均与星形细胞胶质瘤病理分级密切相关(P＜ 0.05).生存素和周期蛋白E阳性共表达率均为50.9%(54/106),两者的表达呈正相关(P＜ 0.01).生存素、周期蛋白E表达与hTP1标记指数有相关性(P＜0.01).结论 生存素、周期蛋白E和hTP1表达与星形细胞胶质瘤恶性程度有关,其表达异常可能在星形细胞胶质瘤的形成和恶变中发挥重要作用.