Targeted inactivation of EPS8 using dendrimer-mediated delivery of RNA interference.

We developed polyamidoamine dendrimers conjugated with epidermal growth factor (EGF) for use in receptor-mediated delivery of therapeutics to cancer cells. Here, we demonstrate the utility of this approach to inhibit proliferation and migration of head and neck squamous carcinoma cells through targeting of EPS8, a key regulator of squamous carcinoma growth and motility. Use of EGF-dendrimers to deliver siRNA or shRNA against EPS8 resulted in inhibition of cell growth and reduction in cell motility. Moreover, more profound repression of the target protein was obtained with repeat exposure to the targeting reagent, and was consistent with the altered biological properties. Thus, targeting of EPS8 can be achieved with EGF-conjugated dendrimers delivering EPS8-specific RNAi therapeutics, leading to a reduction in the malignant phenotype of cells.

CYP1A1 is a target of miR-892a-mediated post-transcriptional repression.

Cytochrome P450 1A1 (CYP1A1) is a member of the cytochrome p450 enzyme family, which is involved in the metabolisms of carcinogenic metabolites, such as benzo(a)pyrene. In this study, we identified miR-892a as a negative regulator of CYP1A1 expression. Luciferase assays revealed a sequence in the 3'-untranslated region of CYP1A1 that displayed a perfect match with miR-892a, and revealed that this sequence was a specific miR-892a target site. The overexpression of miR­892a inhibited the expression of the CYP1A1 protein, and the miR­892a antagonist increased CYP1A1 expression. Of note, benzo(a)pyrene, a major inducer of CYP1A1 transcription, decreased the expression of miR-892a. Moreover, the miR-892a-induced CYP1A1 repression inhibited the benzo(a)pyrene-mediated decrease in cell viability. These data provide insight into the CYP1A1 regulatory network.

Resveratrol alters microRNA expression profiles in A549 human non-small cell lung cancer cells.

Resveratrol is a plant phenolic phytoalexin that has been reported to have antitumor properties in several types of cancers. In particular, several studies have suggested that resveratrol exerts antiproliferative effects against A549 human non-small cell lung cancer cells; however, its mechanism of action remains incompletely understood. Deregulation of microRNAs (miRNAs), a class of small, noncoding, regulatory RNA molecules involved in gene expression, is strongly correlated with lung cancer. In this study, we demonstrated that resveratrol treatment altered miRNA expression in A549 cells. Using microarray analysis, we identified 71 miRNAs exhibiting greater than 2-fold expression changes in resveratrol-treated cells relative to their expression levels in untreated cells. Furthermore, we identified target genes related to apoptosis, cell cycle regulation, cell proliferation, and differentiation using a miRNA target-prediction program. In conclusion, our data demonstrate that resveratrol induces considerable changes in the miRNA expression profiles of A549 cells, suggesting a novel approach for studying the anticancer mechanisms of resveratrol.

Dendrimer-triglycine-EGF nanoparticles for tumor imaging and targeted nucleic acid and drug delivery.

We designed an epidermal growth factor (EGF)-containing polyamidoamine (PAMAM) Generation 4 dendrimer vector labeled with quantum dots for targeted imaging and nucleic acid delivery. (1)H NMR, SDS-PAGE, and Western blotting were applied to characterize the synthesized G4.0-GGG-EGF nanoparticles. Targeting efficiency, cell viability, proliferation, and intracellular signal transduction were evaluated using HN12, NIH3T3, and NIH3T3/EGFR cells. We found that EGF-conjugated dendrimers did not stimulate growth of EGFR-expressing cells at the selected concentration. Consistent with this, minimal stimulation of post-receptor signaling pathways was observed. These nanoparticles can localize within cells that express the EGFR in a receptor-dependent manner, whereas uptake into cells lacking the receptor was low. A well characterized vimentin shRNA (shVIM) and yellow fluorescent protein (YFP) siRNA were used to test the delivery and transfection efficiency of the constructed targeted vector. Significant knockdown of expression was observed, indicating that this vector is useful for introduction of nucleic acids or drugs into cells by a receptor-targeted mechanism.

MicroRNAs that respond to histone deacetylase inhibitor SAHA and p53 in HCT116 human colon carcinoma cells.

MicroRNAs (miRNAs) are important post-transcriptional regulators involved in many biological processes. We investigated the expression profiles of miRNAs affected by the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), and p53 in the human colon cancer cell line, HCT116 (wt-p53) and its derivative, HCT116 (null-p53). In a microarray assay, 144 of 275 human miRNAs showed several-fold changes in transcription. Most of these miRNAs were strongly affected by SAHA, and their expression profiles varied depending on the presence of p53. Thirty-one miRNAs showing the greatest expression changes were selected for target prediction, and genes related to apoptosis (102), cell cycle (38), and differentiation (102) were predicted. Each miRNA had many target genes, and several genes also were targeted by many miRNAs. Putative p53 upstream binding sites for the miRNAs were determined, and most sites scored >85%, suggesting a high probability of binding. In conclusion, we identified several miRNAs whose expression was affected by both SAHA and p53. Many of the miRNAs showed dramatic changes and were predicted to target many mRNAs. Further studies will be needed to verify these predictions.

Alteration of miRNA profiles by ionizing radiation in A549 human non-small cell lung cancer cells.

Ionizing radiation (IR) is widely used in cancer treatment and in biological studies. It disrupts cellular homeostasis through multiple mechanisms including changes of the expression profile of genes. Although microRNAs (miRNAs) have recently been recognized as important post-transcriptional regulators and are involved in various biological processes, whether miRNAs play any roles in the cellular response to IR, is not well examined. We investigated the profile of miRNA expression following IR in the human lung carcinoma cell line A549, and the expression profiles of IR-responsive miRNAs were confirmed by qRT-PCR. The target mRNAs of IR-responsive miRNAs were predicted with a target prediction tool. Microarray analysis identified 12 and 18 miRNAs in 20- and 40 Gy-exposed A549 cells, respectively, that exhibited more than 2-fold changes in their expression levels. Of these, four were changed in only 20-Gy-treated cells, ten only in 40-Gy-treated cells, and eight miRNAs were found to change after both treatments. qRT-PCR analysis of a subset of the miRNAs showed patterns of regulation as the microarray data, although the magnitude of the changes differed in the two data sets. Target prediction for IR-responsive miRNAs suggests that they target genes related to apoptosis, regulation of cell cycle, and DNA damage and repair. Taken together, these data suggest that miRNA expression is affected by radiation, and they may be involved in the regulation of radiation responses.

Suberoylanilide hydroxamic acid (SAHA) changes microRNA expression profiles in A549 human non-small cell lung cancer cells.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor (HDACI) with antitumor effects that is being explored as a therapeutic drug. However, it has been reported that non-small cell lung cancer (NSCLC) is resistant to HDACIs. MicroRNAs (miRNAs) are a key class of small, non-coding RNA molecules that modulate post-transcriptional regulation of gene expression in multi-cellular organisms. miRNA expression patterns are involved in deregulation of gene expression in human lung cancer. Here we identified miRNA expression profile changes in response to SAHA treatment in the human lung carcinoma cell line A549. We also examined potential mRNA targets of SAHA-responsive miRNAs by using a target prediction program. Using microarray analysis, we found 64 miRNAs with >2-fold expression changes in SAHA-treated A549 cells. Among them, two unique miRNAs were altered in 2.5 microM SAHA-treated cells, 31 unique miRNAs were altered in 5.0 microM SAHA-treated cells and 31 miRNAs were altered with both doses. These miRNAs are predicted to have several target genes related to angiogenesis, apoptosis, chromatin modification, cell proliferation and differentiation. In conclusion, we have identified a unique set of miRNAs and their expression profiles that are influenced significantly by SAHA in the A549 NSCLC cell line model, which might provide useful information for understanding the anticancer mechanism of SAHA.

MicroRNAs are significantly influenced by p53 and radiation in HCT116 human colon carcinoma cells.

Ionizing radiation is genotoxic to the cell, and p53 is commonly considered to be a key regulator that controls gene expression responding to the genotoxity of radiation. The expression profiles of microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of target mRNAs, were analyzed to determine whether any correlation exists between miRNA expression, radiation response, and/or p53. The miRNA profiles were analyzed by microarray containing 470 human miRNA probes in HCT116 human colon carcinoma cells and their p53-null derivative. Thirty-eight miRNAs among the 138 flagged human miRNAs were selected by fold-change analysis. The expression levels of these 38 miRNAs were changed more than two-fold, and a total of 12 miRNAs were significantly affected by p53, radiation, and the combination of both. All 12 miRNAs had expression patterns correlated to p53, while two miRNAs were affected by radiation or the combined action of radiation and p53. In bioinformatics studies, these miRNAs had p53-binding sites with scores higher than 85% in their upstream regions, and some of their target genes were found to be involved in genotoxic responses. In conclusion, we have identified miRNAs influenced significantly by p53 and/or radiation in the HCT116 human colon carcinoma cell line model, and these miRNAs may have important roles in the regulation of genes involved the cellular responses to radiation.

Identification of ionizing radiation-responsive microRNAs in the IM9 human B lymphoblastic cell line.

Ionizing radiation (IR) disrupts cellular homeostasis through multiple mechanisms including changes of the expression profile of genes. Although microRNAs (miRNAs), small single-stranded RNAs, have recently been recognized as important post-transcriptional regulators of gene expression, it is not well investigated if miRNAs function in the cellular response to radiation. Therefore, we determined if IR induces changes in the expression profiles of miRNAs and used this approach to identify IR-responsive miRNAs. To monitor the profiles of miRNAs, microarray analysis was conducted with irradiated IM9 human lymphoblastic cells. The expression levels of specific miRNAs were confirmed by quantitative real-time PCR (qRT-PCR) and statistically analyzed. Finally, the target mRNAs of some IR-responsive miRNAs were predicted with two different prediction programs. IR-exposed human lymphoblastic cells underwent cell cycle arrest and apoptosis. Apoptosis was more significantly increased at a higher radiation dose. There were 73 and 33 human miRNAs in 1 and 10 Gy-irradiated cells, respectively that showed expression level changes of >2-fold. By qRT-PCR analysis, it was revealed that the patterns of miRNA expression were similar to those observed in the microarray data, although the quantitative expression levels were discordant. Prediction of genes targeted by IR-responsive miRNA yielded several genes, many of which are involved in the regulation of apoptosis, the cell cycle, and DNA repair. The expression profiles of miRNAs in the IM9 human B lymphoblastic cells are strongly affected by IR and these changes may be involved in the regulation of cellular response to IR.

Regional differences in the prevalence, treatment and control of hypertension and dyslipidemia in US urban Hispanic populations participating in health screening events.

OBJECTIVE: We examine the prevalence, treatment, and control of hypertension, dyslipidemia, and concomitant hypertension and dyslipidemia among Hispanics in four US communities. METHODS: This was a cross-sectional study of Hispanics who participated in health screening programs from 2004 to 2006. We enrolled 5288 Hispanics in Miami (n=372), New York (n=254), Los Angeles (n=4037), and Houston (n=625). The main outcome measures were prevalence, treatment and control rates of hypertension, dyslipidemia, and concomitant hypertension and dyslipidemia. RESULTS: Overall prevalence rates of hypertension, dyslipidemia, and concomitant hypertension and dyslipidemia were 37.5%, 26.6%, and 15.3%, respectively. Hypertension treatment rates ranged from 30.9% (Houston) to 68.2% (Miami) (P<.05); control was achieved in 34.7% (Los Angeles) to 47.8% (New York, P<.05). Dyslipidemia treatment rates were lowest in Houston (36.5%) and highest in New York (75.3%, P<.05); control rates were 62.3% (Houston) to 75.1% (Los Angeles P<.05). Dual treatment of hypertension/dyslipidemia ranged from 24.4% (Houston) to 69.4% (New York, P<.05), dual control was achieved in 4.5% (Houston) to 35.3% (New York, P<.05). Multivariable logistic regression analyses showed the odds of having each condition did not to differ by region, but regional differences existed for treatment and control. CONCLUSIONS: A high prevalence of hypertension, dyslipidemia, and combined hypertension and dyslipidemia and low control rates for hypertension and concomitant hypertension and dyslipidemia exist among US Hispanic adults in different communities.

Efficacy and safety of coadministered amlodipine and atorvastatin in patients with hypertension and dyslipidemia: results of the AVALON trial.

The AVALON study was a randomized, multicenter trial to assess the efficacy and safety of coadministered amlodipine and atorvastatin in patients with hypertension and dyslipidemia. Phase one was an 8-week, double-blind, double-dummy, placebo-controlled period whereby patients received amlodipine 5 mg, atorvastatin 10 mg, amlodipine 5 mg and atorvastatin 10 mg, or placebo. Thereafter, all patients received single-blind amlodipine 5 mg and atorvastatin 10 mg for 8-weeks, followed by 12 weeks of open-label treatment where doses could be titrated to improve low-density lipoprotein cholesterol and blood pressure control. A total of 847 patients entered the double-blind phase. At Week 8, 45% of the patients receiving amlodipine 5 mg and atorvastatin 10 mg reached both their blood pressure and low-density lipoprotein cholesterol goals, compared with 8.3% with amlodipine (p < 0.001), 28.6% with atorvastatin (p < 0.001), and 3.5% with placebo. At 28 weeks, 67.1% of patients coadministered amlodipine and atorvastatin (mean doses, 7.6 mg and 28.4 mg, respectively) achieved both targets. Framingham estimated 10-year risk of coronary heart disease declined from baseline levels of 15.1% to 6.9% at Week 28. Following coadministered treatment, the adverse events reported were similar to either agent alone. Concomitant administration of amlodipine and atorvastatin is an effective and well tolerated treatment for coexisting hypertension and dyslipidemia.