Cancer Cell International - Latest Articles

Localization of CD26/DPPIV in nucleus and its nuclear translocation enhanced by anti-CD26 monoclonal antibody with anti-tumor effect

Kohji Yamada — 2009-06-26T00:00:00Z

Background: CD26 is a type II, cell surface glycoprotein known as dipeptidyl peptidase (DPP) IV. Previous studies have revealed CD26 expression in T cell leukemia/lymphoma and malignant mesothelioma, and an inhibitory effect of anti-CD26 monoclonal antibody (mAb) against the growth of CD26+ cancer cells in vitro and in vivo. The function of CD26 in tumor development is unknown and the machinery with which the CD26 mAb induces its anti-tumor effect remains uncharacterized. Results: The localization of CD26 in the nucleus of T cell leukemia/lymphoma cells and mesothelioma cells was shown by biochemical and immuno-electron microscopic analysis. The DPPIV enzyme activity was revealed in the nuclear fraction of T cell leukemia/lymphoma cells. These expressions of intra-nuclear CD26 were augmented by treatment with the CD26 mAb, 1F7, with anti-tumor effect against the CD26+ T cell leukemia/lymphoma cells. In contrast, the CD26 mAb, 5F8, without anti-tumor effect, did not augment CD26 expressions in the nucleus. Biotin-labeled, cell surface CD26 translocated into the nucleus constantly, and this translocation was enhanced with 1F7 treatment but not with 5F8. Conclusions: These results indicate that the intra-nuclear CD26 which moves from plasma membrane may play certain roles in cell growth of human cancer cells.

Eurycomanone induce apoptosis in HepG2 cells via up regulation of p53

Yusmazura Zakaria — 2009-06-10T00:00:00Z

Background: Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack. Previous studies had noted the cytotoxic effect against various cancer cell lines. The aim of this study is to investigate the cytotoxicity against human hepato carcinoma cell in vitro and the mode of action. The cytotoxicity of eurycomanone was evaluated using MTT assay and the mode of cell death was detected by Hoechst 33258 nuclear staining and flow cytometry with Annexin-V/propidium iodide double staining. The protein expression Bax, Bcl-2, p53 and cytochrome C were studied by flow cytometry using a spesific antibody conjugated fluorescent dye to confirm the up-regulation of p53 and Bax in cancer cells. Results: The findings suggested that eurycomanone was cytotoxic on cancerous liver cell, HepG2 and less toxic on normal cells Chang's liver and WLR-68. Furthermore, various methods proved that apoptosis was the mode of death in eurycomanone-treated HepG2 cells. The characteristics of apoptosis including chromatin condensation, DNA fragmentation and apoptotic bodies were found following eurycomanone treatment. This study also found that apoptotic process triggered by eurycomanone involved the up-regulation of p53 tumor suppressor protein. The up-regulation of p53 was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2. The increased of cytochrome C levels in cytosol also results in induction of apoptosis. Conclusion: The data suggest that eurycomanone was cytotoxic on HepG2 cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2.

Bioluminescence imaging reveals inhibition of tumor cell proliferation by Alzheimer's amyloid beta protein

Hong Zhao — 2009-06-01T00:00:00Z

Background: Cancer and Alzheimer's disease (AD) are two seemingly distinct diseases and rarely occur simultaneously in patients. To explore molecular determinants differentiating pathogenic routes towards AD or cancer, we investigate the role of amyloid β protein (Aβ) on multiple tumor cell lines that are stably expressing luciferase (human glioblastoma U87; human breast adenocarcinoma MDA-MB231; and mouse melanoma B16F). Results: Quantification of the photons emitted from the MDA-MB231 or B16F cells revealed a significant inhibition of cell proliferation by the conditioning media (CM) derived from amyloid precursor protein (APP) over-expressing cells. The inhibition of U87 cells was observed only after the media was conditioned for longer than 2 days with APP over-expressing cells. Conclusion: Our results suggest that Aβ plays an inhibitory role in tumor cell proliferation; this effect could depend on the type of tumor cells and amount of Aβ.

Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

Eugene Fine — 2009-05-29T00:00:00Z

Background: Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration. Methods: Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM), or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2. Results: There was a high correlation of cell growth with ATP concentration (r = 0.948) in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p < 0.05). Conclusion: Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies.

Nucleic acid distribution pattern as a possible biomarker for metabolic activities of neoplastic cells: a digitally-aided fluorescence microscopy study on normal and neoplastic lymphocytes of acute and chronic canine lymphocytic leukemia

Godwin Isitor — 2009-05-11T00:00:00Z

Background: Metabolic states of neoplastic cells are increasingly being relied upon for diagnostic and prognostic assessment of neoplastic conditions. The nucleic acid distribution pattern of cells in general, in terms of degree of condensation of the nuclear chromatin and overall spread of the nucleic acid within the nuclear and cytoplasmic compartments, can reflect the metabolic state of the cell. This simple but logical concept appears not be put into consideration to date as numerous attempts are being made towards formulating reliable biomarkers for rapid diagnosis, prognosis and subsequent therapeutic interventions for neoplastic conditions. We comparatively evaluated nucleic acid distribution patterns of normal lymphocytes and neoplastic cells of lymphocytic lineage, employing light and fluorescence microscopy procedures, as well as digital imaging analytical methods. Results: The results demonstrate distinctiveness in the pattern of nucleic acid distribution for the normal lymphocytes and three lymphocytic neoplastic cell-types of canine lymphocytic leukemia that are categorized as small, intermediate and large neoplastic lymphocytes. Variably-shaped cytoplasmic processes laden with single-stranded nucleic acids (SSNA) were observed for the small and intermediate-sized neoplastic lymphocytes, compared with large neoplastic lymphocytes and the normal lymphocytes; the latter two categories of cells being virtually devoid of similar processes. Prominent cytoplasmic and nuclear clumps of SSNA, indicative of a higher rate of metabolic activity, were also observed within the neoplastic cells compared with fewer and narrower SSNA of the normal cells. Conclusion: The comparative relative increases of SSNA in cytoplasmic processes and other cellular areas of small and intermediate-sized neoplastic lymphocytes is reflective of greater metabolic activity in neoplastic cells in general compared with their normal cellular counterparts.

Gene expression profiling predicts a three-gene expression signature of endometrial adenocarcinoma in a rat model

Sandra Karlsson — 2009-05-08T00:00:00Z

Background: In the Western world, endometrial cancers are the most common gynaecological neoplastic disorders among women. Initial symptoms are often vague and may be confused with several other conditions or disorders. Thus, there is a need for an easy and reliable diagnostic tool. The objective of this work was to identify a gene expression signature specific for endometrial adenocarcinomas to be used for testing potential endometrial biomarkers. Results: Changes in expression between endometrial adenocarcinomas and non-/pre-malignant endometrium from the BDII EAC rat model were compared in cDNA microarray assays. By employing classification analysis (Weka) on the expression data from approximately 5600 cDNA clones and TDT analysis on genotype data, we identified a three-gene signature (Gpx3, Bgn and Tgfb3). An independent analysis of differential expression, revealed a total of 354 cDNA clones with significant changes in expression. Among the 10 best ranked clones, Gpx3, Bgn and Tgfb3 were found. Conclusion: Taken together, we present a unique data set of genes with different expression patterns between EACs and non-/pre-malignant endometrium, and specifically we found three genes that were confirmed in two independent analyses. These three genes are candidates for an EAC signature and further evaluations of their involvement in EAC tumorigenesis will be undertaken.

Down-regulation of SFRP1 in a mammary epithelial cell line promotes the development of a CD44high/CD24low population which is invasive and resistant to anoikis

Kelly Gauger — 2009-05-07T00:00:00Z

Background: The Wnt family of secreted proteins is implicated in the regulation of cell fate during development, as well as in cell proliferation, morphology, and migration. Aberrant activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway by competing with the Frizzled receptor for Wnt ligands resulting in an attenuation of the signal transduction cascade. Loss of SFRP1 expression is observed in breast cancer, along with several other cancers, and is associated with poor patient prognosis. However, it is not clear whether the loss of SFRP1 expression predisposes the mammary gland to tumorigenesis. Results: When SFRP1 is knocked down in a non-malignant immortalized mammary epithelial cell line (76 N TERT), nuclear levels of β-catenin rise and the Wnt pathway is stimulated. The SFRP1 knockdown cells exhibit increased expression of the pro-proliferative Cyclin D1 gene and increased cellular proliferation, undergo a partial epithelial-mesenchymal transition (EMT), are resistant to anchorage-independent cell death, exhibit increased migration, are significantly more invasive, and exhibit a CD24low/CD44high cell surface marker expression pattern. Conclusion: Our study suggests that loss of SFRP1 allows non-malignant cells to acquire characteristics associated with breast cancer cells.

Decytabine enhances cytotoxicity induced by oxaliplatin and 5-fluorouracil in the colorectal cancer cell line Colo-205.

Sylwia Flis — 2009-04-27T00:00:00Z

Background: DNA methylation is an epigenetic phenomenon known to play an important role in the development of cancers, including colorectal cancer (CRC). Aberrant methylation of promoter regions of genes is potentially reversible, and if methylation is important for cancer survival, demethylation should do the opposite. To test this we have addressed the hypothesis that DNA methyltransferase inhibitors (DNMTi), decytabine and zebularine, potentiate inhibitory effects of classical anti-CRC cytostatics, oxaliplatin and 5-fluorouracil (5-FU), on survival of CRC cells in vitro. Results: Isobole and median effect analysis revealed that decytabine shows potent synergistic interaction with oxaliplatin and 5-FU and that this is probably not the class effect of DNMTi as zebularine shows strong antagonistic interaction with oxaliplatin. The synergistic combination treatment was also applied to the cultures to investigate their mechanisms of action. We have shown that combinations of decytabine with cytostatics produced dose-dependent growth inhibition and treatment-induced apoptosis. Conclusion: The observed synergism between decytabine and cytostatics is most probably related to the augmented apoptotic signal and allowed for significant (both biologically and statistically) reduction of the cytotoxic doses of cytostatics used.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Sam-Mui Tsui — 2009-04-17T00:00:00Z

Background: Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable. Methods: The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo. Results: Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared. Conclusion: Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.

Mammaglobin as a potential molecular target for breast cancer drug delivery

Lian Zuo — 2009-03-23T00:00:00Z

Background: Mammaglobin (MAM) has been used as a specific molecular marker for breast cancer diagnosis. Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule. Some of the strategies are based upon an essential but not demonstrated hypothesis – mammaglobin is associated with the surface of breast cancer cells, which strongly disputes the therapeutic strategies. Results: We conducted a computer-based predictive analysis and identified a small fragment at the N-end of MAM as a potential transmembrane domain. We provided several evidences to demonstrate the presence of the membrane-associated MAM. We isolated the membrane protein components from known MAM positive breast cancer cells (MDA-MB361 and MDA-MB415). We showed that about 22–64% of MAM proteins, depending upon the types of the cancer cells, directly attached on the membrane of breast cancer cells, by Western blotting assays. To directly visualize the presence of the membrane-bound MAM protein, we incubated the MAM positive cancer cells with FITC labeled anti-MAM antibody, and observed clear fluorescent signals on the surface of the cells. In studying the MAM protein distribution in human breast cancer tissues, we first identified two immunostain patterns that are associated with the membrane-bound MAM: the membrane stain pattern and luminary surface stain pattern. To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL. Specific binding and cytotoxicity of the MAM targeted and Dox loaded LDL was tested in the MAM positive breast cancer cells in vitro. Conclusion: We first showed that some of MAM protein directly associated with the surface of breast cancer cells. The membrane-associated MAM protein may be utilized as a useful molecular marker for breast cancer targeted drug delivery.