Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Iodide treatment acutely increases pendrin (SLC26A4) mRNA expression in the rat thyroid and the PCCl3 thyroid cell line by transcriptional mechanisms

Iodine is a critical element involved in thyroid hormone synthesis. Its efflux into the follicular lumen is thought to occur, in part, through pendrin at the apical membrane of thyrocytes. This study attempted to investigate whether iodide administration affects SLC26A4 mRNA expression in rat thyroid and in PCCl3 cells. Rats and cells were treated or not with Nal from 30 min up to 48 h. One group was concomitantly treated with sodium perchlorate. SLC26A4 mRNA expression was also investigated in PCCl3 cells treated with actinomycin D prior to Nal treatment. Iodide administration significantly increased SLC26A4 mRNA content in both models. The simultaneous administration of Nal and perchlorate, as well as the treatment of PCCl3 cells with actinomycin D prevented this effect, indicating that intracellular iodide is essential for this event, which appears to be triggered by transcriptional mechanisms. These data show that intracellular iodide rapidly upregulates SLC26A4 mRNA expression. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr(-)) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (similar to 64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditionalmethods of screening high-producing recombinant cellsmay represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

Quantitative correlation between transcriptional levels of ER chaperone, peroximal protein and FVIII productivity in human Hek-293 cell line

Hek-293 cell line presents good production platform for recombinant therapeutic proteins, however little is known about the components that contribute to the cellular control of recombinant protein production. In this study, we generated a Hek-293 producing recombinant factor VIII (FVIII) and we evaluated the immunoglobulin-binding protein (BiP) and phytanoil-CoA α-hydroxylase (PAHX) expression levels which are known for diminishing FVIII production. Our analyses showed that the recombinant cell population expresses 3.1 ± 1.4 fold of BIP mRNA (P = 0.0054) and 97.8 ± 0.5 fold of PAHX mRNA (P = 0.0016) compared to nontransduced cells. The amount of these proteins was inversely correlated to the secreted FVIII. In conclusion, BIP and PAHX expression are augmented in human cells producing FVIII and they antagonize the amount of therapeutic factor VIII in the cell culture.

Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

Abstract Background The CHD7 (Chromodomain Helicase DNA binding protein 7) gene encodes a member of the chromodomain family of ATP-dependent chromatin remodeling enzymes. Mutations in the CHD7 gene are found in individuals with CHARGE, a syndrome characterized by multiple birth malformations in several tissues. CHD7 was identified as a binding partner of PBAF complex (Polybromo and BRG Associated Factor containing complex) playing a central role in the transcriptional reprogramming process associated to the formation of multipotent migratory neural crest, a transient cell population associated with the genesis of various tissues. CHD7 is a large gene containing 38 annotated exons and spanning 200 kb of genomic sequence. Although genes containing such number of exons are expected to have several alternative transcripts, there are very few evidences of alternative transcripts associated to CHD7 to date indicating that alternative splicing associated to this gene is poorly characterized. Findings Here, we report the cloning and characterization by experimental and computational studies of a novel alternative transcript of the human CHD7 (named CHD7 CRA_e), which lacks most of its coding exons. We confirmed by overexpression of CHD7 CRA_e alternative transcript that it is translated into a protein isoform lacking most of the domains displayed by the canonical isoform. Expression of the CHD7 CRA_e transcript was detected in normal liver, in addition to the DU145 human prostate carcinoma cell line from which it was originally isolated. Conclusions Our findings indicate that the splicing event associated to the CHD7 CRA_e alternative transcript is functional. The characterization of the CHD7 CRA_e novel isoform presented here not only sets the basis for more detailed functional studies of this isoform, but, also, contributes to the alternative splicing annotation of the CHD7 gene and the design of future functional studies aimed at the elucidation of the molecular functions of its gene products.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

ORAOV1 is amplified in oral squamous cell carcinoma

BACKGROUND: Oral cancer overexpressed 1 (ORAOV1) was found as a candidate oncogene in the 11q13 chromosomal region, based on its amplification and overexpression in oral cancer cell lines. Because gene amplification often leads to increased levels of gene expression, we aimed to verify the relationship between ORAOV1 gene status and mRNA expression primarily in oral squamous cell carcinoma (OSCC) by quantitative assay, correlating with clinical and pathological characteristics in patients. METHODS: Levels of ORAOV1 amplification and expression were evaluated by qPCR and RT-qPCR in OSCC cell lines and in tumor and non-tumoral surgical margins from 33 patients with OSCC. All subjects were smokers and habitual alcohol drinkers, mostly men above 40 years of age and with a single primary tumor. RESULTS: ORAOV1 exhibited increased gene expression levels as well as higher copy number in three OSCC cell lines with 11q13 amplified chromosomal region when compared with the OSCC cell line without the amplification (one-way ANOVA, P < 0.05). Weak correlation between ORAOV1 mRNA levels and DNA copy number was seen in tumor samples (Spearman, P = 0.07). Although ORAOV1 was amplified in tumor (Wilcoxon, P < 0.01), high levels of transcripts in margin did not reveal differences in comparison with tumor (Wilcoxon, P = 0.85). Aggressiveness and survival rate did not demonstrate statistical difference for both events in OSCC. CONCLUSION: The overexpression of ORAOV1 in non-tumoral margin samples can occur in the absence of amplification. The weak correlation between ORAOV1 amplification and expression in OSSC suggests that ORAOV1 expression can be regulated by mechanisms other than gene amplification. J Oral Pathol Med (2012) 41: 5460

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Oestrogen receptor beta in adenoid cystic carcinoma of salivary glands

Aims: This study aimed to describe the expression of oestrogen receptor (ER)alpha, ER beta and aromatase in salivary gland adenoid cystic carcinoma (ACC). Methods and results: ER alpha, ER beta and aromatase expression was analysed by immunohistochemistry in tissue microarray blocks from 38 cases of ACC and seven normal salivary glands. The intracellular localization and amount of total protein expression were investigated by immunofluorescence and western blotting in an ACC cell line. Western blotting analysis showed overexpression of ER alpha, ER beta and aromatase in the ACC cell line; however, with immunofluorescence, only ER beta was shown to be expressed in the nucleus. Immunohistochemistry revealed positive nuclear expression of ER beta, positive cytoplasmic expression of aromatase and a lack of ER alpha expression as compared with normal salivary glands. Conclusions: The nuclear expression of ER beta indicates that oestrogen may be active in ACC and possibly able to mediate E2-targeted gene transcription. This study strongly suggests that ER beta may be involved in tumour progression, playing a role in tumour development, and thus corroborating the indication for ER antagonists in the clinical control of ACC. This study opens a new perspective on the potential use of anti-oestrogens and aromatase inhibitors as therapeutic agents against ACC.

Cytotoxicity of cashew flavonoids towards malignant cell lines

The leaves of the Cashew plant (Anacardium occidentale L.) are used by the folk medicine in South America and West Africa. This plant is rich in flavonoids, which are polyphenolic compounds widespread in plants, and that have diverse physiological effects. In a sub-acute toxicity assay it was found that an ethanolic extract of Cashew leaves elicited lymphopenia in rats. The extract was also found to be cytotoxic and to induce apoptosis in Jurkat (acute lymphoblastic leukemia) cells. The crude ethanolic extract was fractionated and resolved by HPLC. One of the four fractions obtained led to the isolation of the biflavonoid agasthisflavone. [H-3]-thymidine incorporation assays and flow cytometry analysis showed that the isolated compound displayed a high anti-proliferative effect in Jurkat cells with an IC50 of 2.4 mu g/ml (4.45 mu M). The effect of agathisflavone on the acute promyelocytic leukemia cell line HL60, Burkitt lymphoma Raji cells and Hep-2 laryngeal carcinoma cells was also tested. The two latter ones were only mildly affected by agathisflavone. It is also shown that agathisflavone induces apoptosis in Jurkat cells and it this proposed that this is the likely mechanism of agathisflavone specific cytotoxicity. (C) 2010 Elsevier GmbH. All rights reserved.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. (C) 2012 International Society for Advancement of Cytometry

E series prostaglandins alter the proliferative, apoptotic and migratory properties of T98G human glioma cells in vitro

Background: In many types of cancer, prostaglandin E-2 (PGE(2)) is associated with tumour related processes including proliferation, migration, angiogenesis and apoptosis. However in gliomas the role of this prostanoid is poorly understood. Here, we report on the proliferative, migratory, and apoptotic effects of PGE(1), PGE(2) and Ibuprofen (IBP) observed in the T98G human glioma cell line in vitro. Methods: T98G human glioma cells were treated with IBP, PGE(1) or PGE(2) at varying concentrations for 24-72 hours. Cell proliferation, mitotic index and apoptotic index were determined for each treatment. Caspase-9 and caspase-3 activity was measured using fluorescent probes in live cells (FITC-LEHD-FMK and FITC-DEVD-FMK respectively). The migratory capacity of the cells was quantified using a scratch migration assay and a transwell migration assay. Results: A significant decrease was seen in cell number (54%) in the presence of 50 mu M IBP. Mitotic index and bromodeoxyuridine (BrdU) incorporation were also decreased 57% and 65%, respectively, by IBP. The apoptotic index was increased (167%) and the in situ activity of caspase-9 and caspase-3 was evident in IBP treated cells. The inhibition of COX activity by IBP also caused a significant inhibition of cell migration in the monolayer scratch assay (74%) and the transwell migration assay (36%). In contrast, the presence of exogenous PGE(1) or PGE(2) caused significant increases in cell number (37% PGE(1) and 45% PGE(2)). When mitotic index was measured no change was found for either PG treatment. However, the BrdU incorporation rate was significantly increased by PGE(1) (62%) and to a greater extent by PGE(2) (100%). The apoptotic index was unchanged by exogenous PGs. The addition of exogenous PGs caused an increase in cell migration in the monolayer scratch assay (43% PGE(1) and 44% PGE(2)) and the transwell migration assay (28% PGE(1) and 68% PGE(2)). Conclusions: The present study demonstrated that treatments which alter PGE(1) and PGE(2) metabolism influence the proliferative and apoptotic indices of T98G glioma cells. The migratory capacity of the cells was also significantly affected by the change in prostaglandin metabolism. Modifying PG metabolism remains an interesting target for future studies in gliomas.