Volume 13, Issue 3

Apigenin (4′,5,7,-trihydroxyflavone) is a flavonoid abundant in the common fruits, herbs and vegetables constituting the bulk of the human diet. This study was aimed at quantifying the effects of apigenin on the basic cellular traits determining cancer development, i.e. cell proliferation, gap junctional coupling, and motility, using the Dunning rat prostate MAT-LyLu cell model. We demonstrated that apigenin considerably inhibits MAT-LyLu cell proliferation and significantly enhances the intensity of connexin43-mediated gap junctional coupling. This effect correlates with an increased abundance of C×43-positive plaques at the cell-to-cell borders seen in apigenin-treated variants. Moreover, we observed an inhibitory effect of apigenin on the motility of MAT-LyLu cells. The basic parameters characterising MAT-LyLu cell motility, especially the rate of cell displacement, considerably decreased upon apigenin administration. This in vitro data indicates that apigenin may affect cancer development in general, and prostate carcinogenesis in particular, via its influence on cellular activities decisive for both cancer promotion and progression, including cell proliferation, gap junctional coupling and cell motility and invasiveness.

STGC3 is a novel candidate tumor suppressor gene that was found to be associated with nasopharyngeal carcinoma (NPC) via the cDNA cloning and RACE processes. The biological function of the STGC3 protein and its expression level in nasopharyngeal carcinoma remain unknown. This study aimed to evaluate the STGC3 protein expression level in NPC and to investigate the inhibitory function of STGC3 as a candidate tumor suppressor gene. We assessed the expression of the STGC3 protein in NPC biopsies and normal control specimens via Western blot and immunohistochemical analysis. The expression of STGC3 as induced by doxycycline (Dox) via a tetracycline (Tet)-regulated system in human nasopharyngeal carcinoma cell line CNE2 was also established, and the effect of STGC3 restoration on the biological behavior of CNE2 was observed. A reduced level of STGC3 expression (0.978 ± 0.213 versus 0.324 ± 0.185, P < 0.05) was detected in NPC versus normal nasopharyngeal tissue by Western blot assay. Immunohistochemical assays for STGC3 detected positive staining in the nuclei and cytoplasm of epithelial cells, and the positive expression rate in NPC, 8 of 21 (38%), was lower than that in normal nasopharynx samples, 16 of 22 (72%). After STGC3 expression was restored, the growth capacity and clone formation potential of CNE2 cells in soft agar were significantly suppressed, and the cell percentage in G0/G1 phase increased, while the percentage of cells entering the S and G2 phases decreased. This indicates that an abnormality in STGC3 expression is associated with nasopharyngeal carcinogenesis and that it may play an important role in controlling cell growth and regulating the cell cycle.

The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins, including those that control cell cycle progression, apoptosis, signal transduction and the NF-κB transcriptional pathway. Aberrations in the ubiquitin-proteasome system underlie the pathogenesis of many human diseases, so both the ubiquitin-conjugating system and the 20S proteasome are important targets for drug discovery. This article presents a few of the most important examples of the small molecule inhibitors and modulators targeting the ubiquitin-proteasome system, their mode of action, and their potential therapeutic relevance in the treatment of cancer and inflammatory-related diseases.

This report describes an improvement made to the horizontal cell electrophoresis methodology. It involves using two liquid layers differing in density to produce an interface described as a “density cushion”. The electrophoretic system that employed an anti-convective porous matrix to separate red blood cells (RBC) and charged dyes effectively was found to be unsuitable for some other mammalian cells. The “density cushion” method was found to be more versatile and applicable to studies on the separation of a variety of cell types. The experiments described show the differences between the electrophoretic mobilities of a human eosinophilic leukaemia cell line (Eol-1) and RBC, both with and without the modification of the cell surface properties.

Malignant prostate tissues have markedly reduced zinc (Zn2+) contents in comparison to non-malignant tissues. In this study, we restored a high intracellular Zn2+ level to LNCaP prostate cancer cells by culturing the cells in a growth medium supplemented with a supraphysiological concentration of Zn2+ (10 μg/ml) over 5 weeks. The intracellular Zn2+ level increased in the Zn2+-treated cells, and there was a marked increase in the presence of zincosomes, a Zn2+-specific intracellular organelle. The proliferation rate of the Zn2+-treated cells was markedly reduced. There was also a significant increase (36.6% ± 6.4%) in the total tyrosine phosphorylated proteins. Vaccinia H1-related (VHR) phosphatase, zeta chain-associated protein-70 (ZAP-70) kinase and phosphorylated extracellular signal-regulated protein kinase 1 and 2 (p-ERK 1 and 2) were also present in higher abundance. Treatment with TPEN, which chelates Zn2+, reduced the abundance of VHR phosphatase and ZAP-70 kinase, but increased the abundance of p-ERK 1. However, the TPEN treatment restored the Zn2+-treated LNCaP cell proliferation to a rate comparable to that of the non Zn2+-treated cells. These results highlight the importance of a high intracellular Zn2+ content and the VHR/ZAP-70-associated pathways in the modulation of LNCaP prostate cancer cell growth.

The human ZNF300 gene is a member of the KRAB/C2H2 zinc finger gene family, the members of which are known to be involved in various developmental and pathological processes. Here, we show that the ZNF300 gene encodes a 68-kDa nuclear protein that binds DNA in a sequence-specific manner. The ZNF300 DNA binding site, C(t/a)GGGGG(c/g)G, was defined via a random oligonucleotide selection assay, and the DNA binding site was further confirmed by electrophoretic mobility shift assays. A potential ZNF300 binding site was found in the promoter region of the human IL-2Rβ gene. The results of electrophoretic mobility shift assays indicated that ZNF300 bound to the ZNF300 binding site in the IL-2Rβ promoter in vitro. Transient co-transfection assays showed that ZNF300 could activate the IL-2Rβ promoter, and that the activation was abrogated by the mutation of residues in the ZNF300 binding site. Identifying the DNA binding site and characterizing the transcriptional regulation property of ZNF300 would provide critical insights into its potential as a transcriptional regulator.

p73, a member of the p53 family, exhibits activities similar to those of p53, including the ability to induce growth arrest and apoptosis. p73 influences chemotherapeutic responses in human cancer patients, in association with p53. Alternative splicing of the TP73 gene produces many p73 C- and N-terminal isoforms, which vary in their transcriptional activity towards p53-responsive promoters. In this paper, we show that the C-terminal spliced isoforms of the p73 protein differ in their DNA-binding capacity, but this is not an accurate predictor of transcriptional activity. In different p53-null cell lines, p73β induces either mitochondrial-associated or death receptor-mediated apoptosis, and these differences are reflected in different gene expression profiles. In addition, p73 induces cell cycle arrest and p21WAF1 expression in H1299 cells, but not in Saos-2. This data shows that TAp73 isoforms act differently depending on the tumour cell background, and have important implications for p73-mediated therapeutic responses in individual human cancer patients.

Ionic channels form pores in biomembranes. These pores are large macromolecular structures. Due to thermal fluctuations of countless degrees-of-freedom of the biomembrane material, the actual form of the pores is permanently subject to modification. Furthermore, the arrival of an ion at the binding site can change this form by repolarizing the surrounding aminoacids. In any case the variations of the pore structure are stochastic. In this paper, we discuss the effect of such modifications on the channel conductivity. Applying a simple kinetic description, we show that stochastic variations in channel properties can significantly alter the ionic current, even leading to its substantial increase or decrease for the specific matching of some time-scales of the system.

Membranes made from binary mixtures of egg sphingomyelin (ESM) and cholesterol were investigated using conventional and saturation-recovery EPR observations of the 5-doxylstearic acid spin label (5-SASL). The effects of cholesterol on membrane order and the oxygen transport parameter (bimolecular collision rate of molecular oxygen with the nitroxide spin label) were monitored at the depth of the fifth carbon in fluid- and gel-phase ESM membranes. The saturation-recovery EPR discrimination by oxygen transport (DOT) method allowed the discrimination of the liquid-ordered (l o), liquid-disordered (l d), and solid-ordered (s o) phases because the bimolecular collision rates of the molecular oxygen with the nitroxide spin label differ in these phases. Additionally, oxygen collision rates (the oxygen transport parameter) were obtained in coexisting phases without the need for their separation, which provides information about the internal dynamics of each phase. The addition of cholesterol causes a dramatic decrease in the oxygen transport parameter around the nitroxide moiety of 5-SASL in the l o phase, which at 50 mol% cholesterol becomes ∼5 times smaller than in the pure ESM membrane in the l d phase, and ∼2 times smaller than in the pure ESM membrane in the s o phase. The overall change in the oxygen transport parameter is as large as ∼20-fold. Conventional EPR spectra show that 5-SASL is maximally immobilized at the phase boundary between regions with coexisting l d and l o phases or s o and l o phases and the region with a single l o phase. The obtained results all owed for the construction of a phase diagram for the ESM-cholesterol membrane.

The laminin-binding protein, variously called the 37/67-kDa high affinity laminin receptor or p40, mediates the attachment of normal cells to the laminin network, and also has a role as a ribosomal protein. Over-expression of this protein has been strongly correlated with the metastatic phenotype. However, few studies have investigated the cellular consequence of the ablation of this gene’s expression. To address this issue, the expression of the 37/67-kDa high affinity laminin receptor was knocked out with several siRNA constructs via RNA interference in transformed liver (Hep3B) cells. In each case where the message was specifically ablated, apoptosis was induced, as determined by annexin V/propidium iodide staining, and by double staining with annexin V and an antibody directed against the 37/67-kDa high affinity laminin receptor. These results suggest that this protein plays a critical role in maintaining cell viability.

The relationship between plasma lipid levels and mortality from cardiovascular diseases has been shown in many studies, but there has been far less investigation into their relationship to non-cardiovascular diseases. The aim of this study was to investigate the lipid profile of individuals with hematological malignancies and its relationship to disease activity. 238 patients were included in the study: 84 with acute leukemia, 62 with non-Hodgkin lymphoma, 35 with Hodgkin’s lymphoma, 32 with multiple myeloma, and 25 with myeloproliferative syndrome. The HDL cholesterol level of the patients differed to that of the individuals in the control group in the active disease period for all the analyzed disorders, but only remained statistically significant in the acute leukemia and non-Hodgkin lymphoma groups during the remission period. Smaller differences were observed for the remaining lipid fractions, except for the triglyceride level, which increased in the active disease period in all the analyzed disorders except non-Hodgkin lymphoma. The most pronounced changes in the lipid fractions occurred in the HDL cholesterol level, and were the most remarkable for acute leukemia.

Ionizing radiation is one of the types of oxidative stress that has a number of damaging effects on cutaneous tissues. One of the histological features of radiation-induced cutaneous fibrosis is the accumulation of extracellular matrix (ECM) components, including heparan sulfate proteoglycan (HSPG), which are required for the repair of tissue damage, and operate by interacting with a variety of growth factors. In this study, we established a model of human HaCaT keratinocytes overexpressing anti-oxidative enzyme genes to elucidate the mechanism of oxidative stress leading to the accumulation of HSPG and the role of its accumulation. Catalase overexpression induced an increase in anti-HS antibody (10E4) epitope expression in these cells. Western blotting showed that the smeared bands of HSPG were obviously shifted to a higher molecular weight in the catalase transfectants due to glycosylation. After heparitinase I treatment, the core proteins of HSPG were expressed in the catalase transfectants to almost the same extent as in the control cells. In addition, the transcript levels of all the enzymes required for the synthesis of the heparan sulfate chain were estimated in the catalase transfectant clones. The levels of five enzyme transcripts — xylosyltransferase-II (XT-II), EXTL2, D-glucuronyl C5-epimerase (GLCE), HS2-O-sulfotransferase (HS2ST), and HS6-O-sulfotransferase (HS6ST) — were significantly increased in the transfectants. Moreover, hydrogen peroxide was found to down-regulate the levels of these enzymes. By contrast, siRNA-mediated repression of catalase decreased 10E4 epitope expression, the transcript level of HS2ST1, and the growth rate of HaCaT cells. These findings suggested that peroxide-mediated transcriptional regulation of HS metabolism-related genes modified the HS chains in the HaCaT keratinocytes.