HAI-1过表达对SW620细胞体外生长和运动能力的影响

肝细胞生长因子激活因子抑制因子1(hepatocyte growth factor activator inhibitor type 1, HAI-1)能有效抑制肝细胞生长因子激活因子(hepatocyte growth factor activator,HGFA)和丝氨酸蛋白酶Matriptase的活性,并可通过对HGFA和Matriptase活性的调控参与HGF/c-Met信号传导途径。为了解HAL-1在肿瘤细胞的生长和运动中的作用,本研究将人HAI-1基因全长cDNA克隆至pcDNA3.1( )表达载体,并转染人肠癌SW620细胞,用Western blot验证了转染细胞中HAI-1的表达情况,并分别利用生长曲线、软琼脂集落形成、穿膜运动和扩散运动测定等方法检测了HAI-1 过表达对SW620细胞生长和运动能力的影响。生长曲线和软琼脂集落形成测定都显示出HAI-1 转染细胞与对照组相比差异不十分明显。穿膜运动和扩散运动测定则均显示了HAI-1过表达对细胞运动能力有明显的抑制。因此,HAI-1的过表达虽然在体外对肿瘤细胞生长影响较小,但可以抑制肿瘤细胞的运动迁移能力。     

人组氨酸磷酸酶蛋白PHP14的过表达及其对NIH-3T3细胞体外增殖和非锚定依赖性生长的影响

目的:获取人组氨酸磷酸酶蛋白PHP14基因,并构建其N端和C端GFP融合的真核表达载体,建立过表达PHP14的NIH-3T3细胞系,并观察其对NIH-3T3细胞体外增殖和非锚定依赖性生长的影响.方法:以HeLa cDNA为模板,PCR扩增PHP14的全长编码基因,分别克隆到pEGFP-N2和pEGFP-C3载体中,构建pEGFP-N2-PHP14和pEGFP-C3-PHP14真核表达载体,利用脂质体将构建的载体转染到NIH-3T3细胞中,MTT法检测细胞增殖,软琼脂成集落法检测体外非锚定依赖性生长能力.结果:成功构建了过表达PHP14的真核表达载体pEGFP-N2-PHP14和pEGFP-C3-PHP14,并在NIH-3T3细胞中检测到了目的基因的过表达,PHP14在NIH-3T3细胞中过表达并不影响NIH-3T3细胞的体外增殖,但赋予了NIH-3T3细胞非锚定依赖性生长的能力.结论:成功构建了过表达PHP14的NIH-3T3细胞模型,在NIH-3T3细胞中过表达PHP14并不影响NIH-3T3细胞的体外增殖,但赋予了NIH-3T3细胞非锚定依赖性生长能力.     

Checkpoint kinase 1 modulates sensitivity to cisplatin after spindle checkpoint activation in SW620 cells

Aneuploidy is a common feature of tumours that arise by errors in chromosome segregation during mitosis. The aim of this study was to evaluate possible signaling pathways involved in sensitization to chemotherapy in cells with chromosomal instability. We designed a screen using the fission yeast Squizossaccharomyces pombe, to isolate strains showing a phenotype of chromosome mis-segregation and higher sensitivity to the antitumoral drug Bleomycin. We examined differences in gene expression using a comparative analysis of genome-wide expression of the wild type strain and one of the mutants. The results revealed a set of genes involved in cell cycle control, including Mad3/BubR1 and Chk1. We then studied the levels of these two proteins in colorectal cancer human cell lines with different genomic content. Among these, SW620 cells showed higher BubR1 and Chk1 mRNA levels than control cells under normal conditions. Since Chk1 is required for both S and G2/M checkpoints, and the microtubule-destabilizing agent, nocodazole induces mitotic arrest, we attempted to investigate the potential anticancer effects of nocodazole in combination with cisplatin. These studies showed that SW620 cells undergo synergistic cell death after spindle checkpoint activation followed by cisplatin treatment, suggesting a role of Chk1 in this checkpoint, very likely dependent on BubR1 protein. Importantly, Chk1-depleted SW620 cells lost this synergistic effect. In summary, we propose that Chk1 could be a biomarker predictive of the efficacy of chemotherapy across different types of tumors with aneuploidy. These findings may be potentially very useful for the stratification of patients for treatment.     

Resveratrol induces mitochondrial respiration and apoptosis in SW620 colon cancer cells

BackgroundThe polyphenol resveratrol (RSV) is found in the skin of red grapes and has been reported to exhibit anticancer properties. The antitumor effects of RSV in the gastrointestinal tract have gained considerable interest due to the high exposure of this tissue to this dietary compound. One of the hallmarks of cancer cells is their particular metabolism mainly relying on glycolysis for ATP production rather than mitochondrial oxidative phosphorylation. Although RSV has been described to act as a calorie-restriction mimetic, modulating energy metabolism in normal tissues, little efforts have been done to study the effects of this polyphenol in the metabolism of cancer cells. Taking this into account, the aim of this study was to explore metabolic effects of this polyphenol in colon cancer.MethodsOxygen consumption, ATP levels, Western blotting and other molecular biology techniques were carried out to characterize the metabolic signature of RSV in SW620 colon cancer cells.ResultsParadoxically, the cytotoxic effects of RSV were associated with an increase in oxygen consumption supported by mitochondrial biogenesis and increased fatty acid oxidation. This partial reversion of the Warburg effect was followed by hyperpolarization of mitochondrial membrane and ROS production, leading to an increased apoptosis.ConclusionsOur results propose that the anticancer mechanisms of RSV could reside in targeting cancer cell metabolism, promoting mitochondrial electron transport chain overload and, ultimately, increasing ROS production.General significanceThese results shed new light into the anticancer mechanism of RSV supporting the ability of this compound in potentiating the effects of chemotherapy.     

Effect of alpha-fetoprotein on the growth of human hepatoma cells in vitro]

The stimulatory activity of human alpha-fetoprotein (AFP) on the growth of mouse hepatoma-22 cells had been reported in our previous paper. The present work aimed at further investigation of the effect of AFP on human hepatoma cell growth by MTT colorimetric assay. The results showed that AFP could stimulate the growth of SMMC-7721 human hepatoma cells in vitro. The present results also showed that the stimulatory effect of AFP on the growth of SMMC-7721 cells was decreased by the anti-serum of AFP. The anti-AFP antibody alone could suppress the growth of SMMC-7721 cells. On the other hand, AFP and anti-AFP antibody had no effect on the growth of HL-60 human leukemia cells, indicating that the tumor cell growth stimulating effect of AFP was not simply due to non-specific addition of exogenous protein and this effect of AFP showed strict tumor cell specificity. In addition, MCF-7 human breast cancer cell growth was also promoted by AFP and inhibited by anti-AFP antibody. Because AFP cell-surface receptors have been detected in MCF-7 breast cancer cells, and AFP could also be produced and secreted by MCF-7 cells, the possibility may be considered: AFP may bind with its receptors on tumor cell membrane for the purposes of growth stimulation.     

Neuregulin-1 enhances motility and migration of human astrocytic glioma cells

Gliomas are the most frequently diagnosed adult primary brain malignancy. These tumors have a tendency to invade diffusely into the surrounding healthy brain tissue, thereby precluding their successful surgical removal. In this report, we examine the potential for the neuregulin-1/erbB receptor signaling network to contribute to this process by modulating glioma cell motility. Neuregulin-1 is expressed throughout the immature and adult central nervous system and has been demonstrated to influence the migration of a variety of cell types in the developing brain. In addition, erbB2, an integral member of the heterodimeric neuregulin-1 receptor, has been shown to be overexpressed in human glioma biopsies. Using antibodies specific for erbB2 and erbB3, we show that these receptors localize preferentially in regions of the plasma membrane which are involved in facilitating cellular movement. Here, erbB2 colocalizes and coimmunoprecipitates with members of the focal complex including beta1-integrin and focal adhesion kinase. Further, erbB receptor activation by neuregulin-1 enhances cell motility in two-dimensional scratch motility assays and stimulates cell invasion in three-dimensional Transwell migration assays. These effects of neuregulin-1 appear to involve the activation of focal adhesion kinase, which occurs downstream from erbB2 receptor stimulation. Taken together these data suggest that neuregulin-1 plays an important modulatory role in glioma cell invasion.     

SW620细胞RACK1稳定RNA干扰细胞系的构建与鉴定

目的:为研究RACK1在结肠癌发生发展中的作用,构建结肠癌RACK1基因稳定RNA干扰(RNAi)细胞系。方法:根据人Gnb211 c DNA序列,运用干扰原则选择5个干扰位点并合成相应干扰片段,定向克隆入p Lentilox3.7干扰载体鼠U6启动子后并测序验证。用干扰及对照质粒分别转染HEK293T细胞48小时后,RT-PCR鉴定干扰效率,选出干扰效率较高的质粒包装慢病毒感染人结肠癌细胞SW620,流式无菌分选出荧光阳性的细胞扩增培养,RT-PCR及Western blotting鉴定慢病毒干扰效率。使用慢病毒构建的SW620 RACK1稳定RNAi细胞系及对照组进行MTT实验初步研究RACK1对SW620增殖的影响。结果:酶切和测序证实RACK1sh RNA质粒构建正确,产生能同时表达绿色荧光蛋白(EGFP)和RACK1 sh RNA的慢病毒载体质粒。慢病毒转导SW620并流式无菌分选扩增培养后,与空载体组相比,2个RNAi组均不同程度抑制RACK1表达,RACK1sh RNA5抑制作用最明显,RACK1干扰组细胞增殖得到了抑制。结论:SW620细胞RACK1稳定RNAi细胞系构建成功,为深入研究RACK1在结直肠癌发生发展中的作用奠定了基础。     

Proteins excreted by primary human colon carcinoma cells (SW 480) promote spreading and growth of metastatic human colon carcinoma cells (SW 620) in serum-free medium

The growth behavior of the two human colon tumor cell lines (SW 480, primary and SW 620, metastatic), originating from the same patient, was studied in six different serum-free media (SFM) [GF3, Chee's essential medium plus insulin, transferrin and selenium; GF3F, GF3 plus fetuin; GF4, GF3 plus linoleic acid-BSA; GF5, GF4 plus fetuin; GF5E, GF5 plus EGF; GF5T, GF5 plus triiodothyronine]. SW 480 grew in all of the SFM. In contrast, SW 620 grew only in four SFM. The cells did not grow in GF3 and GF4. When grown in SFM, SW 480 attached much more firmly to the dishes than SW 620 as determined by the time required to detach the cells with trypsin-EDTA (SW 480, greater than 20 min and SW 620, less than 5 min). It was speculated that SW 480 cells excrete proteins in SFM which influence attachment and growth of the cells. Growth behavior of SW 480 cells which did not grow in GF3, was studied using GF3 medium and SW 480 substratum dishes. SW 620 cells readily attached to the SW 480 substratum dishes and grew. Furthermore, when SW 620 cells were grown on substratum prepared from serum-supplemented medium incubated in the absence of cells (serum substratum), the cell growth was comparable to the cell growth on SW 480 substratum in GF3. Substratum from SW 480 cells and the serum substratum were compared for their components using SDS-PAGE system. The SW 480 substratum contains many more components than serum substratum. A protein band at 60 kD appears to be common in both SW 480 and serum substrata.     

Cooperative Anti-Invasive Effect of Cdc42/Rac1 Activation and ROCK Inhibition in SW620 Colorectal Cancer Cells with Elevated Blebbing Activity

Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.     

Effect of DOPA and dopamine on the motility of isolated rat duodenum in vitro]

The motor effects of DOPA and Dopamine on the isolated rat duodenum in vitro have been studied by establishing successive dose- response curves. These effects are either excitatory or inhibitory according to the concentrations used. In every case they are of small amplitude. The inhibitory effects do not exist in the presence of alpha, and beta blocking agents. The excitatory effects are suppressed by using a serotoninergic blocking agent.