Ectopic expression of the ErbB-3 binding protein ebp1 inhibits growth and induces differentiation of human breast cancer cell lines

Ebp1, an ErbB-3 binding protein, translocates from the cytoplasm to the nucleus of human breast cancer cells after treatment with the ErbB-3 ligand, heregulin. The purpose of these studies was to examine the effects of ectopic expression of ebp1 on the biological properties of human ErbB-3-expressing breast carcinoma cell lines. Ectopic expression of ebp1 in ErbB-2, ErbB-3-expressing breast carcinoma cell lines resulted in inhibition of colony formation, a decreased proliferation rate, an accumulation of cells in the G2/M phase of the cell cycle, and suppression of growth in soft agar. Ectopic expression of ebp1 led to a more differentiated phenotype in AU565 breast cancer cells, as evidenced by increased expression of lipid droplets and of the milk protein casein. Basal phosphorylation of extracellular regulated kinases (Erks) 1 and 2, kinases activated by heregulin treatment, was also observed in ebp1 transfectants. The promoter for the intercellular adhesion molecule-1 gene, a heregulin-inducible gene, was constitutively activated in ebp1 transfectants as determined by reporter construct analysis. These data demonstrate that ectopic expression of the ErbB-3 binding protein Ebp1 inhibits proliferation and induces differentiation of ErbB-2, ErbB-3-expressing human breast carcinoma cell lines.     

Gastrin inhibits motility,decreases cell death levels and increases proliferation in human glioblastoma cell lines

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 373–382, 1998     

Protein-tyrosine kinase and protein-serine/threonine kinase expression in human gastric cancer cell lines

Protein kinases play key roles in cellular functions. They are involved in many cellular functions including; signal transduction, cell cycle regulation, cell division, and cell differentiation. Alterations of protein kinase by gene amplification, mutation or viral factors often induce tumor formation and tumor progression toward malignancy. The identification and cloning of kinase genes can provide a better understanding of the mechanisms of tumorigenesis as well as diagnostic tools for tumor staging. In this study, we have used degenerated polymerase-chain-reaction primers according to the consensus catalytic domain motifs to amplify protein kinase genes (protein-tyrosine kinase, PTK, and protein-serine/threonine kinase, PSK) from human stomach cancer cells. Following amplification, the protein kinase molecules expressed in the gastric cancer cells were cloned into plasmid vectors for cloning and sequencing. Sequence analysis of polymerase-chain-reaction products resulted in the identification of 25 protein kinases, including two novel ones. Expression of several relevant PTK/PSK genes in gastric cancer cells and tissues was further substantiated by RT-PCR using gene-specific primers. The identification of protein kinases expressed or activated in the gastric cancer cells provide the framework to understand the oncogenic process of stomach cancer.     

Culture filtrates of Aspergillus fumigatus induce different modes of cell death in human cancer cell lines

Aspergillus fumigatus culture filtrate (CF) has a potent cytotoxic effect on three human cancer cell lines (DLKP, A549 and HEp-2) and initiates cell death by apoptosis but the execution of the apoptotic process is incomplete. DLKP cells treated with A. fumigatus CF demonstrate features associated with apoptosis but cytoplasmic and nuclear fragmentation were not observed and cells ultimately underwent necrosis. The apoptotic process commenced in A549 and HEp-2 cells upon exposure to CF, cell shrinkage was observed but membrane blebbing and apoptotic body formation were not detected and detached cells died by necrosis. In contrast, extensive nuclear fragmentation and apoptotic body formation were evident in DLKP and A549 cells treated with anti-neoplastic agents. This work indicates that A. fumigatus CF is cytotoxic to cancer cells and can initiate apoptosis but that the complete apoptotic pathway is not followed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line

Attrora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma （ESCC）. It has been demonstrated that cells overexpressing Attrora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Attrora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly （ADPribose） polymerase （PARP） in Attrora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A＇s effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.     

Pharmacological eEF2K activation promotes cell death and inhibits cancer progression

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV‐PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti‐viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir‐resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir‐mediated anti‐tumoral activity is severely compromised in eEF2K‐deficient engrafted tumors in vivo. Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti‐tumoral properties of HIV‐PIs.     

Downregulation of RPL6 by siRNA inhibits proliferation and cell cycle progression of human gastric cancer cell lines

Our previous study revealed that human ribosomal protein L6 (RPL6) was up-regulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer from drug-induced apoptosis. It was further demonstrated that up-regulation of RPL6 accelerated growth and enhanced in vitro colony forming ability of GES cells while down-regulation of RPL6 exhibited the opposite results. The present study was designed to investigate the potential role of RPL6 in therapy of gastric cancer for clinic. The expression of RPL6 and cyclin E in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemisty. It was found that RPL6 and cyclin E were expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa and the two were correlative in gastric cancer. Survival time of postoperative patients was analyzed by Kaplan- Meier analysis and it was found that patients with RPL6 positive expression showed shorter survival time than patients that with RPL6 negative expression. RPL6 was then genetically down-regulated in gastric cancer SGC7901 and AGS cell lines by siRNA. It was demonstrated that down-regulation of RPL6 reduced colony forming ability of gastric cancer cells in vitro and reduced cell growth in vivo. Moreover, down-regulation of RPL6 could suppress G1 to S phase transition in these cells. Further, we evidenced that RPL6 siRNA down-regulated cyclin E expression in SGC7901 and AGS cells. Taken together, these data suggested that RPL6 was over-expressed in human gastric tissues and caused poor prognosis. Down-regulation of RPL6 could suppress cell growth and cell cycle progression at least through down-regulating cyclin E and which might be used as a novel approach to gastric cancer therapy.     

Depletion of Jab1 inhibits proliferation of pancreatic cancer cell lines

Jab1 overexpression is observed in many human cancers, but its physiological significance remains to be investigated. We reduced the level of Jab1 expression in pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 by the RNA interference and found that Jab1-knockdown resulted in impaired cell proliferation and enhanced apoptosis regardless of the genotype of the tumor suppressor p53. This growth inhibition was rescued by the introduction of siRNA-resistant mouse Jab1 cDNA. Jab1-knocked-down cells expressed a higher level of c-myc, and additional depletion of c-myc rescued cells from Jab1-knockdown-mediated growth suppression. Thus, Jab1 overexpression contributes to pancreatic cancer cell proliferation and survival. Jab1 could be a novel target in cancer therapy.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.     

CD151蛋白促内皮细胞增殖和eNOS蛋白表达

目的研究CD151及其突变体CD151-ARSA245-248对人脐静脉内皮细胞(HUVEC)增殖及eNOS表达的影响,探讨CD151促血管生成的机制。方法构建pAAV-CD151及其突变体CD151-ARSA245-248(囊泡运输缺陷突变体),并转染HU-VEC。CCK-8法测定HUVEC增殖的能力,Western Blot检测CD151及eNOS蛋白的表达。结果 pAAV-CD151组及pAAV-CD151-ARSA245-248组CD151蛋白表达均增加,显著高于正常对照组和pAAV-GFP组(P<0.05),但pAAV-CD151组及pAAV-CD151-ARSA245-248组之间CD151蛋白表达没有统计学意义(P>0.05)。CCK-8法测定HUVEC增殖能力亦无统计学意义(P>0.05)。正常对照组,pAAV-GFP组,pAAV-CD151组及pAAV-CD151-ARSA245-248突变体组的OD值分别为1.393?.685、1.498?.746、2.346?.52和1.71?.863,pAAV-CD151组较pAAV-GFP组和正常对照组细胞增殖能力明显增强(P<0.01),pAAV-CD151-ARSA245-248组较pAAV-CD151组细胞增殖能力减弱(P<0.05)。此外,pAAV-CD151组eNOS蛋白表达较pAAV-GFP组和正常对照组明显增加(P<0.01),pAAV-CD151-ARSA245-248组较pAAV-CD151组eNOS蛋白表达降低(P<0.05)。结论 CD151是促细胞增殖的重要蛋白质,CD151影响eNOS信号通路的激活。上述机制可能为CD151促血管生成的重要机制之一。     

Trichosanthin inhibits breast cancer cell proliferation in both cell lines and nude mice by promotion of apoptosis

Breast cancer ranks as a common and severe neoplasia in women with increasing incidence as well as high risk of metastasis and relapse. Translational and laboratory-based clinical investigations of new/novel drugs are in progress. Medicinal plants are rich sources of biologically active natural products for drug development. The 27-kDa trichosanthin (TCS) is a ribosome inactivating protein purified from tubers of the Chinese herbal plant Trichosanthes kirilowii Maximowicz (common name Tian Hua Fen). In this study, we extended the potential medicinal applications of TCS from HIV, ferticide, hydatidiform moles, invasive moles, to breast cancer. We found that TCS manifested anti-proliferative and apoptosis-inducing activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cells. Flow cytometric analysis disclosed that TCS induced cell cycle arrest. Further studies revealed that TCS-induced tumor cell apoptosis was attributed to activation of both caspase-8 and caspase-9 regulated pathways. The subsequent events including caspase-3 activation, and increased PARP cleavage. With regard to cell morphology, stereotypical apoptotic features were observed. Moreover, in comparison with control, TCS- treated nude mice bearing MDA-MB-231 xenograft tumors exhibited significantly reduced tumor volume and tumor weight, due to the potent effect of TCS on tumor cell apoptosis as determined by the increase of caspase-3 activation, PARP cleavage, and DNA fragmentation using immunohistochemistry. Considering the clinical efficacy and relative safety of TCS on other human diseases, this work opens up new therapeutic avenues for patients with estrogen-dependent and/or estrogen-independent breast cancers.     

Cyclin-dependent kinase inhibitor 3 is overexpressed in hepatocellular carcinoma and promotes tumor cell proliferation

Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatases family and has a dual function in cell cycling. The function of this gene has been studied in several kinds of cancers, but its role in human hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that CDKN3 was frequently overexpressed in both HCC cell lines and clinical samples, and this overexpression was correlated with poor tumor differentiation and advanced tumor stage. Functional studies showed that overexpression of CDKN3 could promote cell proliferation by stimulating G1-S transition but has no impact on cell apoptosis and invasion. Microarray-based co-expression analysis identified a total of 61 genes co-expressed with CDKN3, with most of them involved in cell proliferation, and BIRC5 was located at the center of CDKN3 co-expression network. These results suggest that CDKN3 acts as an oncogene in human hepatocellular carcinoma and antagonism of CDKN3 may be of interest for the treatment of HCC.     

Endonuclease G promotes cell death of non-invasive human breast cancer cells

The invasiveness of breast cancer cells was shown to be associated with the suppressed ability to develop apoptosis. The role of cell death DNases/endonucleases has not been previously examined in relation with the invasiveness of breast cancer cells. We have compared the activity of the endonucleases in seven human breast cancer cell lines different in the level of invasiveness and differentiation. The invasiveness of cell lines was confirmed by an in vitro Matrigel-based assay. The total endonuclease activity in the differentiated non-invasive (WDNI) cell lines was higher than that in the poorly differentiated invasive (PDI) cells. The expression of EndoG strongly correlated with the degree of estrogen receptor expression and showed an inverse correlation with vimentin and matrix metalloproteinase-13. The EndoG-positive WDNI cells were more sensitive to etoposide- or camptothecin-induced cell death than EndoG-negative PDI cells. Silencing of EndoG caused inhibited of SK-BR-3 WDNI cell death induced by etoposide. Human ductal carcinomas in situ expressed high levels of EndoG, while invasive medullar and ductal carcinomas had significantly decreased expression of EndoG. This correlated with decreased apoptosis as measured by TUNEL assay. Our findings suggest that the presence of EndoG in non-invasive breast cancer cells determines their sensitivity to apoptosis, which may be taken into consideration for developing the chemotherapeutic strategy for cancer treatment.