Rapamycin-induced cytotoxic signal transduction pathway

We examined the effects of rapamycin on activation, proliferation, and expression of cytotoxic effector molecules in Molt-4 human T lymphocytes. We investigated the effects of rapamycin on cell viability, caspase family protein activities. Western blots of Bcl-2, Bak, p53, p21, p27, Rb, CDK2, and cyclin B1, as well as measurement of reactive oxygen species (ROS) generation and mitochondrial membrane potential transition. Cells were cultured in the presence or absence of rapamycin. Flow cytometric analysis was performed using propidium iodide stain. Viability of Molt-4 cells was decreased by the addition of rapamycin in dose- and time-dependent manners. Rapamycin induced no nuclear fragmentation in Molt-4 cells. Generation of H₂O₂ in rapamycin-treated Molt-4 cells increased in a time-dependent manner. There were no changes among catalytic activities of caspase proteases. And there was no evidence of expression of Bcl-2, p53, p21, p27, or Rb proteins. G2/M phase cell cycle arrest was identified by flow cytometry. We noted decreased expressions of CDK2 and cyclin B1. We also noted increased Bak protein expression and change in mitochondrial membrane potential transition. In conclusion, rapamycin-induced cytotoxicity was characterized by generation of ROS, which modulated Bak protein expression and mitochondrial dysfunction. G2/M phase cell cycle arrest was achieved by decreased expressions of CDK2 and cyclin B1.     

Biomarkers for predicting the response of esophageal squamous cell carcinoma to neoadjuvant chemoradiation therapy

This review summarizes and evaluates the literature regarding the biomarkers for predicting the response and/or prognosis of esophageal squamous cell carcinoma (ESCC) patients treated with neoadjuvant chemoradiation therapy (CRT). There are seven categories of molecules known to correlate with the response and/or prognosis: tumor suppressors (p53, p21), cell cycle regulators (Cyclin D1, CDC25B, 14-3-3sigma), DNA repair molecules (p53R2, ERCC1), drug resistance proteins [metallothionein (MT)], angiogenic factors (VEGF), molecules involved in cell proliferation/invasion/metastasis (Ki-67, COX-2) and hedgehog signaling molecules (Gli-1). Of the above molecules, the tumor suppressor p53 is expected to be a representative biomarker for predicting the response and prognosis. The cell cycle markers CDC25B and 14-3-3sigma have potential as response biomarkers independent of the p53 status. The DNA repair markers, p53R2 or ERCC1, angiogenic molecule (VEGF), and hedgehog signaling pathway factor Gli-1 also have potential to predict the response and prognosis of ESCC. However, there are still many unanswered questions with regard to predicting the clinical effects of neoadjuvant CRT.     

P53 pathways involving G2 checkpoint regulators and the role of their subcellular localisation

DNA damage activates checkpoint pathways to produce a G1 or G2 cell cycle arrest and DNA repair. G2 checkpoint integrity prevents inappropriate mitosis of unrepaired DNA. Cell cycle progression is determined by cyclin-dependent kinase (CDK) enzymes in association with specific cyclin proteins, with Cdc2/cyclin B regulating mitosis. The tumour suppressor p53 re-enforces G2 arrest through the CDK inhibitor, p21(WAF1/CIPI). Functional regulation of G2 checkpoint proteins occurs through levels of protein expression, phosphorylation and subcellular localisation     

Capsaicin induces cycle arrest by inhibiting cyclin‐dependent‐kinase in bladder carcinoma cells

Objective: Capsaicin is a specialized agonist of transient receptor potential vanilloid type 1 Ca2⁺ channel, a member of the vanilloid receptor family of cation channels. We aimed to investigate the effects of capsaicin on the proliferation and cell death of human bladder cancer cells. Methods: Human bladder cancer cell line 5637 was cultured and the expression of transient receptor potential vanilloid type 1 verified by immunofluorescence and Western blot. Cells were given different disposals (different capsaicin concentration with/without pre‐treating with capsazepine; capsazepine, acting as a competitive antagonist of capsaicin) to observe cell viability, cell cycle and cell death by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay and flow cytometry. The apoptosis indexes, such as intracellular production of reactive oxygen species and mitochondrial membrane potential were assessed to elucidate the potential mechanism of capsaicin effects in the cells. Results: Capsaicin decreased the viability of 5637 cells in a dose‐dependent way. The flow cytometry outcome showed that capsaicin blocked the cell cycle in the G0/G1 period. The Western blot of cyclin‐dependent‐kinase involved in G1/S transfer verified this. Meanwhile, increased reactive oxygen species production and decreased mitochondrial membrane potential were detected in capsaicin‐treated groups. Conclusions: Capsaicin induces cell death through increased reactive oxygen species and decreased mitochondrial membrane potential. Furthermore, capsaicin inhibits the proliferation of 5637 bladder carcinoma cells by cycle arrest with the inhibition of CDK2, CDK4 and CDK6.     

Prominent induction of cyclin B1 in G2/M renal cancer cells with butyrolactone 1

BACKGROUND: Butyrolactone 1 (BL) is a cyclin dependent kinase (CDK) inhibitor derived from Aspergillus terreus. None of the present drugs are effective for the treatment of renal cell carcinoma. The use of BL is expected to promote a new type therapy of renal cancer. METHODS: We investigated three human renal cancer cell lines: ACHN, OS-RC-2 and RCC10RGB, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and two-color flow cytometry. Simultaneous measurements of DNA content and cyclin expression allowed us to perform cell cycle specific analysis. Western blot analysis was performed using ACHN to represent cell lines. RESULTS: BL inhibited cell proliferation and caused cell accumulation at G2/M phase associated with the emergence of the third peak. Moreover, BL induced cyclin B1 over-expression in G2/M cells. These changes were quite definite, whereas cyclins D1, E and A showed no changes at all. Cyclin B1 accumulation was confirmed by western blot analysis. The chronological observation revealed that the emergence of the third peak preceded the regression of the increased cyclin B1 positive G2/M cells. These results suggested that BL accelerated cyclin B1 accumulation in G2/M cells, which then shifted to G1 phase without cell division. New G1 cells started DNA synthesis most likely as endoreduplication to form the third peak and the mechanism of cyclin B1 accumulation converted into down-regulation. CONCLUSION: BL induced significant cell kinetic interference in the tested human renal carcinoma cell lines. This might indicate the possibility of a new medical treatment modality for renal cancer.     

强脉冲光对长波紫外线Ⅰ诱导成纤维细胞光损伤的保护作用

目的 研究强脉冲光（intense pulse light,IPL）对长波紫外线（ultraviolet A,UVA Ⅰ）诱导的正常离体人皮肤成纤维细胞（fibroblast,FB）损伤的保护作用,探讨以IPL为手段的光子嫩肤技术的理论基础.方法 分离并培养人FB,用不同剂量UVA Ⅰ照射FB以诱导细胞光损伤,CCK-8检测其增殖能力的情况,根据预实验结果确定IPL剂量进行照射,流式细胞仪技术枪测细胞周期,Western印迹法检测CylinD1和CDK2蛋白的表达水平.结果 不同剂量的UVA Ⅰ可造成FB的损伤,随着UVA Ⅰ剂量的增加,细胞增殖能力下降,11 J/cm2的剂量可明显导致细胞大量死亡,而7 J/cm2的UVAⅠ对细胞的增殖能力没有明显的影响;经UVA Ⅰ照射2 d后再进行IPL连续照射2 d,细胞增殖活性高于单独UVA Ⅰ处理组,差异具有统计学意义.流式细胞仪检测结果表明该组细胞增殖指数升高.细胞周期蛋白CyclinD1和CDK2的表达水平升高.结论 IPL可通过调节周期蛋白的表达而促进正常FB增殖,从而保护UVAⅠ诱导的FB光损伤,为应用IPL面部美容除皱即光子嫩肤术提供理论依据.     

Melatonin inhibits the proliferation of human osteosarcoma cell line MG-63

It seems established that the onset of osteosarcoma and the reduction in melatonin production run in parallel; this suggests that the decline in the cancer-inhibiting agent, melatonin, may contribute to the occurrence of osteosarcoma and that melatonin supplementation may have promise for preventing the development and progression of this condition. There is, however, no direct evidence regarding an antiproliferative effect of melatonin in osteosarcoma cells. In the current study, we examined whether melatonin inhibits the proliferation of human osteosarcoma cell line MG-63. MTT staining showed that at 4 mM–10 mM concentrations, melatonin significantly reduced the MG-63 cell proliferation in a dose-dependent and time-dependent manner. Flow cytometry documented that 4 mM melatonin significantly increased the fraction of cells in the G₀/G₁ phase of the cell cycle, while simultaneously reducing the proportion in the S and G₂/M phases. Western blot and real-time PCR analyses further confirmed that melatonin's inhibitory effect was possibly because of downregulation of cyclin D1 and CDK4, related to the G₁ phase, and of cyclin B1 and CDK1, related to the G₂/M phase. There was no downregulation of cyclin E, CDK2, and cyclin A, which are related to G₁/S transition and S phase. These findings provide evidence that melatonin may significantly inhibit human osteosarcoma cell proliferation in a dose-dependent and time-dependent manner and this inhibition involves the downregulation of cyclin D1, CDK4, cyclin B1 and CDK1.     

Antiproliferative and apoptotic effects of rofecoxib on esophageal cancer in vitro(1)

BACKGROUND: The incidence of Barrett's adenocarcinoma has increased dramatically in the United States, whereas squamous cell carcinoma of the esophagus remains a worldwide problem. Cyclooxygenase (COX)-2 may play an important role in gastrointestinal carcinogenesis and is overexpressed in both Barrett's metaplasia and adenocarcinoma. We hypothesized that a selective and commercially available COX-2 inhibitor, rofecoxib (Vioxx), would inhibit growth of Barrett's adenocarcinoma and squamous cell carcinoma of the esophagus by apoptotic pathways. Additional comparison studies were performed with commercially available COX-2 and COX-1 inhibitors. MATERIALS AND METHODS: Two esophageal adenocarcinoma cell lines (SEG-1 and BIC) and two esophageal squamous cell cancer lines (KYSE 150 and KYSE 410) were treated with rofecoxib at doses ranging from 8.0 to 125 microg/well. NS-398 (a COX-2 antagonist) and Catechin (a COX-1 antagonist) were also used at doses of 50 and 100 microM. Esophageal cell viability was measured by MTT at 24 and 72 h. Apoptosis was evaluated after 18 h of incubation with rofecoxib, NS398, and Catechin by flow cytometry via annexin V assay. RESULTS: Rofecoxib, NS-398, and Catechin treatments all resulted in significant antiproliferative effects in both adenocarcinoma and squamous cell carcinoma of the esophagus in vitro. Substantial increases in apoptotic activity were also found in all cell lines. CONCLUSIONS: Our findings suggest that COX-2 and COX-1 inhibition has potential to become an effective treatment for both histological variants of esophageal cancer. Further in vivo and human studies are warranted to evaluate the safety and clinical utility of these agents in patients with all cancers of the esophagus.     

Effect of cysteine-rich protein 61 on proliferation and cell cycle in human renal tubular epithelial cells

Objective To investigate the effect of cysteine-rich protein 61 (Cyr61) on proliferation and cell cycle in human renal tubular epithelial cells (HK-2). Methods Cyr61 cDNA was cloned into pEGFP-N2, then HK-2 cells were transfected with the recombinant plasmid pEGFP-N2-Cyr61 by Lipofectamine. The cell proliferation was measured by MTT. The expression level of Cyr61, p-FAK and cyclin dependent cyclin-dependent kinase 2 (CDK2) protein were detected by Western blotting. The cell cycle and cell apoptosis were analyzed by flow cytometry. Results The recombinant plasmid pEGFP-N2-Cyr61 could be transfected into HK-2 efficiently. After transfection, the proliferative activity was significantly increased, the proportion of HK-2 cells in G1 phase decreased and in S-phase increased significantly, the level of cell apoptosis decreased markedly (all P＜0.01). The expressions of Cyr61, p-FAK and CDK2 in Cyr61-transfected group were all amplified significantly (all P＜0.01). Conclusions Cyr61 protein over-expressed in HK-2 cells can increase CDK2 expression throngh FAK pathway, resulting in the promotion of HK-2 cells entering into S phase, cell proliferation and the reduction of cell apoptosis.     

Third place--Resident Research Competition, AAO-2000. Antisense cyclin D1 inhibits growth of head and neck cancer xenografts in nude mice.

PROBLEM: Cyclin D1 is a regulatory factor essential in the progression of the cell cycle from G1 through S phase. Amplification and overexpression of cyclin D1 have been observed in many human cancers including head and neck squamous cell carcinoma (HNSCC). We have previously transfected a HNSCC control cell line (CCL23) with an antisense cyclin D1 plasmid and demonstrated inhibition of cell proliferation in vitro. In this study, we examine whether antisense cyclin D1 could inhibit tumor growth in vivo. Methods/measures: The CCL23 and its antisense cyclin D1 transfected clone (CCL23 AS) were injected into the flanks of nude mice. Tumor growth was monitored weekly. After 5 weeks, tumors were removed and studied for tumor size, cyclin D1 expression, cyclin D1-dependent kinase activity, and retinoblastoma (Rb) phosphorylation. RESULTS: Compared with the control tumors, 11 of 19 antisense tumors were smaller, 7 tumors were of equal size, and 1 tumor was larger. Immunohistochemical analysis with an anti-cyclin D1 antibody demonstrated decreased cyclin D1 expression in CCL23 AS and the smaller antisense tumors. Cyclin D1-dependent kinase activity was reduced in CCL23 AS and the smaller antisense tumors, and this was accompanied by a relative decrease in phosphorylated Rb in these samples. CONCLUSION: Antisense cyclin D1 inhibits growth of HNSCC tumors. Cyclin D1 expression, cyclin D1-dependent kinase activity, and Rb phosphorylation are decreased in these tumors. Clinical significance: These findings lend support for the potential use of antisense cyclin D1 as gene therapy for HNSCC.     

Estrogen and Progestin Regulation of Cell Cycle Progression

Estrogens and progesterone, acting via theirspecific nuclear receptors, are essential for normalmammary gland development and differentiated function.The molecular mechanisms through which these effects are mediated are not well defined, althoughsignificant recent progress has been made in linkingsteroid hormone action to cell cycle progression. Thisreview summarizes data identifying c-myc and cyclin D1 as major downstream targets of bothestrogenand progestin-stimulated cell cycle progressionin human breast cancer cells. Additionally, estrogeninduces the formation of high specific activity forms of the cyclin E-Cdk2 enzyme complex lacking thecyclin-dependent kinase (CDK)3 inhibitor, p21. Thedelayed growth inhibitory effects of progestins, whichare likely to be prerequisites for manifestation of their function in differentiation, alsoinvolve decreases in cyclin D1 and E gene expression andrecruitment of CDK inhibitors into cyclin D1-Cdk4 andcyclin E-Cdk2 complexes. Thus estrogens and progestins affect CDK function not only by effects oncyclin abundance but also by regulating the recruitmentof CDK inhibitors and, as yet undefined, additionalcomponents which determine the activity of the CDK complexes. These effects of estrogens andprogestins are likely to be major contributors to theirregulation of mammary epithelial cell proliferation anddifferentiation.     

CO2气腹对胃癌细胞周期及细胞周期蛋白的影响

目的 通过检测不同压力CO2气腹环境下胃癌细胞周期及其相关蛋白表达的变化,探讨CO2气腹对胃癌细胞生长增殖的影响.方法 将胃癌MKN-45细胞置于0、10、12和15 mm Hg CO2气腹环境下培养4 h,然后用流式细胞法检测胃癌细胞周期的变化,再用Western blot方法检测细胞周期蛋白Cyclin D1、CDK4、Rb和pRb的表达,最后用免疫沉淀法检测细胞Cyclin D1/CDK4结合力.结果 0、10、12 mm Hg CO2气腹组胃癌细胞增殖指数与对照组比较差异均无统计学意义(P>0.05),15 mm Hg CO2气腹组胃癌细胞增殖指数(27.4%4±3.7%)与对照组(36.4%±3.3%)比较显著下降(P<0.05).0、10、12 mm Hg CO2气腹组CDK4、Cyclin D1、pRb蛋白表达以及Cyclin D1和CDK4的结合能力与对照组比较差异均无统计学意义(P>0.05),15 mm Hg CO2气腹组CDK4、Cyclin D1、pRb蛋白表达以及Cyclin D1和CDK4的结合能力(0.71%±0.12%、0.93%±0.21%、0.54%±0.11%、0.18%±0.02%)与对照组(1.05%±0.16%、1.40%±0.24%、0.75%±0.14%、0.31%±0.02%)比较显著下降(P<0.05).0、10、12、15 mm Hg CO2气腹组Rb蛋白表达与对照组比较差异无统计学意义(P>0.05).结论 临床常用压力的CO2气腹对胃癌细胞周期无显著影响,15 mm Hg CO2气腹抑制细胞增殖,其原因可能与Cyclin D1和CDK4的表达下调有关.     

细胞周期素E及细胞周期素依赖性激酶2在胃癌组织中的表达及意义

目的探讨胃癌组织中细胞周期素E(cyclinE)及细胞周期素依赖性激酶2(CDK     

TRIM55对大鼠系膜细胞增殖的调控作用

目的系膜增殖性肾炎是世界范围内高发的肾小球疾病,其发病与系膜细胞异常增殖有关,但调控系膜细胞增殖的内在分子机制尚不明确。本研究旨在探索TRIM55对大鼠系膜细胞(RMCs)增殖的调控作用及机制。 方法向8周龄雄性SD大鼠尾静脉注射2.5 mg/kg抗Thy-1抗体建立抗Thy-1肾炎模型。PAS染色观察肾脏病理表现,qPCR检测大鼠肾小球TRIM55 mRNA表达量;利用质粒及siRNA转染分别得到TRIM55过表达及低表达的RMCs,利用流式细胞仪检测其细胞周期;Western印迹检测p27及Cyclin D1的蛋白表达量。 结果TRIM55在抗Thy-1肾炎模型系膜增殖期高表达(P<0.01,T＝3.625)。体外RMCs中,TRIM55过表达可促进RMCs细胞周期由G1期向G2/S期转化,诱导系膜细胞增殖(P<0.01,T＝13.1);TRIM55低表达可引起RMCs的G1期阻滞,抑制RMCs增殖(P<0.01,T＝5.31)。此外,TRIM55过表达可下调p27表达、上调Cyclin D1;反之,TRIM55低表达可上调p27表达、下调Cyclin D1。 结论TRIM55可通过调节p27及Cyclin D1表达水平影响细胞由G1期到G2/S期的转化,进而调控RMCs的增殖。     

中药癌痛克对人胃癌细胞SGC-7901增殖、凋亡作用及机制研究

目的通过观察不同浓度癌痛克对胃癌细胞株SGC-7901增殖及凋亡的作用，以及在相应状态下细胞内视网膜母细胞瘤（Rb）基因相对表达量的改变，探讨中药癌痛克的抗胃癌作用机制。方法以不同浓度癌痛克作用SGC-7901细胞，CCK-8检测细胞增殖抑制率，流式细胞术检测细胞凋亡率细胞周期，RT—PCR方法检测细胞内Bcl-2基因表达变化。结果2～50μg／ml癌痛克可抑制SGC-7901细胞增殖并诱导其凋亡，其抑制及诱导凋亡效应呈浓度依赖性；细胞周期分析处于G1期细胞百分比明显增多，处于G2／M期的细胞减少，Rb基因mRNA表达上调。结论中药癌痛克可通过抑制SGC-7901细胞增殖或诱导其凋亡，且其机制可能通过上调Rb基因表达途径使细胞阻滞在G1期，进而诱导细胞凋亡。     

17-丙烯胺-17-去甲氧格尔德霉素对胆管癌细胞株QBC939生长周期及凋亡的影响

目的 观察热休克蛋白90(HSP 90)抑制剂17-丙烯胺-17-去甲氧格尔德霉素(17-AAG)对胆管癌细胞生长的影响.方法 应用噻唑蓝(MTT)比色法观察17-AAG对胆管癌细胞系QBC939细胞的生长抑制作用;用流式细胞术分析细胞凋亡及细胞周期变化;瑞特吉姆染色法观察细胞形态学;细胞免疫组织化学检测药物处理前后QBC939细胞CDK1和C-e-rbB2的表达变化.结果 17-AAG呈时间、剂量依赖性抑制QBC939细胞,48 h半数抑制浓度(IC50)为1.0umol/L;经17-AAG作用36 h后细胞阻滞于G2期从(11.90±0.78)%上升到(40.33±0.91)%,伴有S期细胞减少;17-AAG处理48 h后早期凋亡明显增加;同时17-AAG处理后的CDK1和C-erbB-2的表达明显减少.结论 HSP90抑制剂17-AAG通过抑制HSP90的分子伴侣功能,下调CDK1和C-erbB2表达,从而抑制细胞增殖,诱导细胞凋亡和周期阻滞.