Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

Eag1 channels as potential early-stage biomarkers of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide. HCC is usually asymptomatic at potential curative stages, and it has very poor prognosis if detected later. Thus, the identification of early biomarkers and novel therapies is essential to improve HCC patient survival. Ion channels have been proposed as potential tumor markers and therapeutic targets for several cancers including HCC. Especially, the ether à-go-go-1 (Eag1) voltage-gated potassium channel has been suggested as an early marker for HCC. Eag1 is overexpressed during HCC development from the cirrhotic and the preneoplastic lesions preceding HCC in a rat model. The channel is also overexpressed in human HCC. Astemizole has gained great interest as a potential anticancer drug because it targets several proteins involved in cancer including Eag1. Actually, in vivo studies have shown that astemizole may have clinical utility for HCC prevention and treatment. Here, we will review first some general aspects of HCC including the current biomarkers and therapies, and then we will focus on Eag1 channels as promising tools in the early diagnosis of HCC.

Posttranscriptional regulation of the ß2-subunit of cardiac L-type Ca2+ channels by MicroRNAs during long-term exposure to isoproterenol in rats.

INTRODUCTION AND METHODS: The effects of long-term ß-adrenergic administration on the expression levels of the cardiac L-type Ca channel ß2 subunit, which regulates channel trafficking and function, were characterized in adult rats. RESULTS: Systemic administration of isoproterenol (150 mg·kg·h) for 2 d led to a 50% increase in the ventricular wet weight-to-body weight ratio (mg/g) and of more than two-fold in the expression of actin protein. In contrast, ß2 subunit protein levels decreased (down to 49%), while mRNA levels remained unchanged. Furthermore, levels of microRNAs (miRs), including miR-21 and miR-132, were upregulated (7.2 and 7.9 fold, respectively). Transfection of these miRs into HEK293 cells attenuated expression of a luciferase reporter gene controlled by a conserved 3'-untranslated region (UTR) of the ß2 subunit (down to 67% and 56%, respectively). Systemic administration of isoproterenol also led to briefer intracellular Ca transients during action potentials measured in isolated cardiomyocytes (down to 65%). CONCLUSION: These results suggest that cardiac L-type Ca channel ß2 subunit protein expression may be downregulated by miRs in response to long-term activation of ß-adrenergic signaling, possibly as an adaptive response in cardiac hypertrophy and sustained ß-adrenergic states.

Pharmacokinetic properties of pravastatin in Mexicans: An open-label study in healthy adult volunteers.

BACKGROUND: The pharmacokinetic properties of pravastatin, particularlyAUC and Cmax, are variable by population. A description of the pharmacokinetic properties of pravastatin in Mexican mestizos was not found in a search of MEDLINE/PubMed (key terms: pravastatin, Mexican, and pharmacokinetics; years: 1966-2005). Because Mexicans and Japanese have common ancestors (Mongoloid group), they also have a common gene pool. This gene pool was modified by genetic "bottlenecks" that occurred when these populations migrated to the Americas and when the Mexican population mixed with the Spanish population during the 16th and 17th centuries. Previous studies in Japanese subjects showed 5 main mutations on the hepatic drug transporter OATP-C, resulting in higher Cmax and AUC values compared with whites. In the Japanese population, the rates of expression of the (*) 1b and (*) 15 alleles were 46% and 15%, respectively. OBJECTIVE: The aim of this study was to evaluate the pharmacokinetic propertiesof pravastatin in healthy Mexican mestizo volunteers and to compare them with those in white and Japanese populations described in the literature. METHODS: This open-label, uncontrolled pilot study of the pharmacokineticproperties of pravastatin was conducted at the Division of Pharmacology, Center for Research and Advanced Studies, Mexico City, Mexico. Healthy, adult, Mexican volunteers received a single dose of pravastatin 10 mg PO (tablet). High-performance liquid chromatography was used to determine plasma pravastatin concentrations between 15 minutes and 12 hours after dosing. RESULTS: Twenty-four subjects (15 women, 9 men; mean age, 30.6 years)participated in the study. The mean (SD) Cmax was 9.5 (2.4) ng/mL; Tmax, 0.8 (0.3) hours; AUC0-∞ 35.7 (19.7) ng/mL - h; t1/2, 2.7 (1.1) hours; and mean residence time, 3.1 (1.1) hours. One volunteer (4%) had an AUC value that differed substantially from the rest of the study population, producing a bimodal distribution of the pharmacokinetic parameters. No adverse events were observed or reported during the trial. CONCLUSIONS: In this small pilot study of the pharmacokinetic properties of pravastatin in Mexican mestizos, AUC was not statistically significantly different from previous studies, either in a white or Japanese population. However, we did not find the high values reported for Cmax in some Japanese subjects carrying recently reported mutations on the pravastatin transporter.

Pharmacokinetics of ciprofloxacin in healthy Mexican volunteers.

Ciprofloxacin (CAS 85721-33-1) is a gyrase inhibitor used against a wide number of bacteria which develops a very low bacterial resistance. Its pharmacokinetics has been extensively studied using two main methods, liquid chromatography and microbiological assay. The pharmacokinetics of ciprofloxacin administered orally was evaluated in healthy Mexican volunteers after a dose of 500 mg. Ciprofloxacin was assayed from plasma by a specific HPLC method reading absorbance at 280 nm. Pharmacokinetic parameters were similar to reported values, including a previous study on Mexican healthy volunteers with the microbiological assay. AUC was 12.11 mg h/l, Cmax 2.44 mg/l, tmax 0.79 h and half-life 3.8 h. tmax was slightly shorter than those in other studies. Ciprofloxacin presented no adverse reactions and can be dosed to people with Amerindian origin in the same dose regimes as prescribed to Caucasians in order to achieve minimal inhibitory concentrations against a wide range of microbial pathogens.

Kupffer cells are responsible for liver cirrhosis induced by carbon tetrachloride.

Inhibition of Kupffer cells could disrupt the sequence of events leading to organ injury by damping down the fibrogenic stimulus. To elucidate the role of Kupffer cells in liver fibrosis and cirrhosis, rats were treated with gadolinium chloride (GdCl(3)) and cirrhosis was induced by subchronic carbon tetrachoride (CCl(4)) administration. Carbon tetrachloride was administered three times per week for 8 weeks to male Wistar rats treated simultaneously with GdCl(3) (20 mg kg(-1), i.p. daily); appropriate controls were performed. Serum enzyme activities of alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GTP) and alanine aminotransferase (ALT) and bilirubin concentration increased significantly by CCl(4), whereas GdCl(3) prevented completely the increase in gamma-GTP and partially prevented the increase in ALP, ALT and bilirubins (P < 0.05). Liver glycogen was depleted by CCl(4), an effect that GdCl(3) was not capable of preventing. Moreover, gadolinium by itself depleted it. Lipid peroxidation increased about 2.5-fold by administration with CCl(4), whereas GdCl(3) preserved lipid peroxidation within normal values. Hepatic collagen increased threefold after subchronic intoxication with CCl(4) (P < 0.05) whereas GdCl(3) prevented partially (P < 0.05) the increase in collagen content, as evidenced by the liver hydroxyproline content and by the histopathological analysis. The present results suggest that Kupffer cells are needed for the production of CCl(4)-induced cirrhosis, because their inactivation with GdCl(3) prevents the disease.