Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 aminoacids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressorgene, with possible relevance for glioblastoma therapy. (C) 2009 Elsevier Ltd. All rights reserved.

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: Structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1–4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1–4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6–7) and tributyltin(IV) complexes (8–11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1–3 are also comparable to those of its o-analogs i.e. 4–7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41–53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1–3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8–11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1–7 and tributyltin(IV) complexes 8–11 is also discussed against a panel of human tumor cell lines.

Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

Alkaloids extracted from Pterogyne nitens induce apoptosis in malignant breast cell line

In the present study, two alkaloids isolated from Pterogyne nitens, a plant native to Brazil, have been shown to induce apoptosis in human breast cancer cells. These compounds, pterogynine (PGN) and pterogynidine (PGD), were tested for their effect on a human infiltrating ductal carcinoma cell line (ZR-7531). The cell line was treated with each alkaloid at several concentrations. Time-dependence (with or without recuperation time) and concentration-dependence (in the range 0.25-10 mM) were investigated in cytotoxicity and apoptosis assays. The annexin assay indicated an apparently higher percentage of death by necrosis of malignant cells after 24 h exposure to both P. nitens extracts than the Hoechst assay. Thus, our results in the two tests demonstrated that the Hoechst assay can discriminate between late apoptotic cells and necrosis, whereas the flow cytometry-based annexin V assay cannot. We concluded that PGN and PGD have effective antineoplastic activity against human breast cancer cells in vitro, by inducing programmed cell death.

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.

Hypusine Modification of the Ribosome-binding Protein eIF5A, a Target for New Anti-Inflammatory Drugs: Understanding the Action of the Inhibitor GC7 on a Murine Macrophage Cell Line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antitumor activity of irradiated riboflavin on human renal carcinoma cell line 786-O

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

Identification of cadmium-induced mutations in a mammalian cell line

Epidemiological studies have shown cadmium to induce cancer in humans, while experimental studies have proven this metal to be a potent tumor inducer in animals. However, cadmium appears nonmutagenic in most prokaryotic and eukaryotic mutagenesis assays. In this study, we present the identification of mutations in normal rat kidney cells infected with the mutant MuSVts110 retrovirus (6m2 cells) as a result of treatment with cadmium chloride. The detection of these mutations was facilitated by the use of a novel mutagenesis assay established in this laboratory. The 6m2 reversion assay is a positive selection system based on the conditional expression of the MuSVts110 v-mos gene. In MuSVts110 the gag and mos genes are fused out of frame, thus the translation of the v-mos sequence requires a frameshift in the genomic RNA. In 6m2 cells this frameshift is accomplished by the temperature-dependent splicing of the primary MuSVts110 transcript. Splicing of MuSVts110, which is mediated by cis-acting sequences, occurs when 6m2 cells are grown at 33$\sp\circ$C and below, but not at 39$\sp\circ$C. Therefore, 6m2 cells appear transformed at low growth temperatures, but take on a morphologically normal appearance when grown at high temperatures. The treatment of 6m2 cells with cadmium chloride resulted in the outgrowth of a number of cells that reverted to the transformed state at high growth temperatures. Analysis of the viral proteins expressed in these cadmium-induced 6m2 revertants suggested that they contained mutations in their MuSVts110 DNA. Sequencing of the viral DNA from three revertants that constitutively expressed the P85$\sp{gag{-}mos}$ transforming protein revealed five different mutations. The Cd-B2 revertant contained three of those mutations: an A-to-G transition 48 bases downstream of the MuSVts110 3$\sp\prime$ splice site, plus a G-to-T and an A-to-T transversion 84 and 100 bases downstream of the 5$\sp\prime$ splice site, respectively. The Cd-15-5 revertant also contained a point mutation, a T-to-C transition 46 bases downstream of the 5$\sp\prime$ splice site, while Cd-10-5 contained a three base deletion of MuSVts110 11 bases upstream of the 3$\sp\prime$ splice site. A fourth revertant, Cd-10, expressed a P100$\sp{gag{-}mos}$ transforming protein, and was found to have a two base deletion. This deletion accomplished the frameshift necessary for v-mos expression, but did not alter MuSVts110 RNA splicing and the expression of p85$\sp{gag{-}mos}.$ Lastly, sequencing of the MuSVts110 DNA from three spontaneous revertants revealed the same G to T transversion in each one. This was the same mutation that was found in the Cd-B2 revertant. These findings provide the first example of mutations resulting from exposure to cadmium and suggest, by the difference in each mutation, the complexity of the mechanism utilized by cadmium to induce DNA damage. ^

*Ras proteins and human tumor cell radiosensitivity

Ras proteins (H-, N-, K4A-, and K4B) are associated with cellular resistance to ionizing radiation (IR) and, consequently, may provide a potential target for radiosensitization strategies in cancer treatment. Several approaches have been used to compromise Ras activity and enhance IR-induced cell killing; however, these techniques either target proteins in addition to Ras or only target one member of the Ras family. In this study, I have used an adenovirus (AV1Y28) that expresses a single-chain antibody fragment directed against Ras proteins to investigate the mechanism(s) responsible for Ras-mediated radiation resistance. AV1Y28 enhanced the radiosensitivity of a number of human tumor cell lines without affecting the radiosensitivity of normal human fibroblasts. Whereas AV1Y28-mediated sensitization was independent of ras gene mutational status, it was dependent on active Ras proteins suggesting that AV1Y28 may be useful against a broad range of tumors. AV1Y28-mediated cell killing was not the result of redistributing cells into a more radiosensitive phase of the cell cycle and did not enhance IR-induced apoptosis. Given that Ras proteins transduce environmental signals to the nucleus, the effect of AV1Y28 on the IR-inducible transcription factor NF-κB were determined. Although AV1Y28 inhibited IR-induced NF-κB through the suppression of IKK, additional work established that NF-κB did not play a role in AV1Y28-mediated radiosensitization. However, a novel component of the signaling pathway responsible for IR-induced NF-κB was identified. Previous studies had suggested a relationship between mutant ras genes and IR-induced G2 delay; therefore the effects of AV1Y28 on the progression of cells from G2 to M after IR were determined. Pretreatment of cells with AV1Y28 prevented the IR-induced G2 arrest. AV1Y28-mediated abrogation of IR-induced G2 arrest correlated with those cell line lines that were sensitized by AV1Y28. Moreover, a significant increase in cells undergoing mitotic catastrophe was found after IR in AV1Y28 treated cells. The abrogation of G2 arrest by AV1Y28 was the result of maintaining the active form of cdc2, an inducer of mitosis, after exposure to IR. This study identified the mechanism of AV1Y28-mediated radiosensitization and has provided insight into the signal transduction pathways responsible for Ras-mediated radiation resistance. ^

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library

Cancer/testis (CT) antigens—immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types—are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5′ end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor)

The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the in vivo growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.