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.

Expression of KATP channels in human cervical cancer: Potential tools for diagnosis and therapy.

Various ion channels, including ATP-sensitive potassium (KATP) channels, are expressed in cancer and have been suggested as potential tumor markers and therapeutic targets. KATP channels are composed of at least two types of subunit, an inwardly rectifying K+ channel (Kir6.x) and a sulfonylurea receptor (SUR). However, the association between KATP channels and cervical cancer remains elusive. The present study determined that the Kir6.2, SUR1 and SUR2 subunits are expressed in cervical cancer cell lines and/or human biopsies. The potential association of subunit expression with tumor differentiation and invasion was analyzed. The effect of the KATP channel blocker glibenclamide on the proliferation of cervical cancer cell lines was also studied. Five cervical cancer cell lines, two primary cultures of cervical cancer cells, one normal keratinocyte cell line and 74 human biopsies were used in the experiments. The mRNA and protein levels of the Kir6.2 subunit were assessed by reverse transcription-polymerase chain reaction and immunochemistry, respectively. Cell proliferation was evaluated by MTT assay. Kir6.2 subunit overexpression compared with control, was observed in some cervical cancer cell lines and cervical tumor tissues. Additionally, increased KATP channel expression was observed in high-grade, poorly differentiated and invasive human cervical cancer biopsies. Kir6.2 subunit expression was not observed in the majority of the non-cancerous cervical tissues. The effect of the KATP channel blocker glibenclamide on the proliferation of five different cervical cancer cell lines was studied, revealing that as Kir6.2 mRNA expression increased, the inhibitory effect of glibenclamide also increased. The results of the present study suggest, for the first time to the best of our knowledge, that the KATP channel subunits, Kir6.2 and SUR2, could potentially represent tools for diagnosing and treating cervical cancer.

Calcium-activated potassium channels as potential early markers of human cervical cancer.

Cervical cancer is a major cause of cancer-associated mortality in women in developing countries. Thus, novel early markers are required. Ion channels have gained great interest as tumor markers, including cervical cancer. The calcium-activated potassium channel KCNMA1 (subunit α-1 from subfamily M) has been associated with different malignancies, including tumors such as breast and ovarian cancer that are influenced by hormones. The KCNMA1 channel blocker iberiotoxin decreases the proliferation of HeLa cervical cancer cells. Nevertheless, KCNMA1 channel expression during cervical carcinogenesis remains elusive. Therefore, KCNMA1 expression was studied in cervical cancer development. FVB transgenic mice expressing the E7-oncogene of high-risk human papilloma virus, and non-transgenic mice were treated with estradiol-releasing pellets during 3 or 6 months to induce cervical lesions. Twenty-four human cervical biopsies from non-cancerous, low- or high-grade intraepithelial lesions, or cervical cancer were also studied. mRNA and protein expression was assessed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. Cervical dysplasia and carcinoma were observed only in the transgenic mice treated with estradiol for 3 and 6 months, respectively. Estradiol treatment increased KCNMA1 mRNA and protein expression in all groups; however, the highest levels were observed in the transgenic mice with carcinoma. KCNMA1 protein expression in the squamous cells of the transformation zone was observed only in the transgenic mice with cervical dysplasia or cancer. Human biopsies from non-cancerous cervix did not display KCNMA1 protein expression; in contrast, the majority of the tissues with cervical lesions (16/18) displayed KCNMA1 protein expression. The lowest channel immunostaining intensity was observed in biopsies from low-grade dysplasia and the strongest in the carcinoma tissues. These results suggest KCNMA1 channels as potential early cervical cancer markers.

The combination astemizole-gefitinib as a potential therapy for human lung cancer.

Lung cancer is a major cause of cancer mortality. Thus, novel therapies are urgently needed. Repositioning of old drugs is gaining great interest in cancer treatment. Astemizole is an antihistamine proposed to be repositioned for cancer therapy. This drug targets several molecules involved in cancer including histamine receptors, ABC transporters and the potassium channels Eag1 and HERG. Astemizole inhibits the proliferation of different cancer cells including those from cervix, breast, leukemia and liver. Gefitinib is widely used to treat lung cancer; however, no response or drug resistance occurs in many cases. Here, we studied the combined effect of astemizole and gefitinib on the proliferation, survival, apoptosis and gene and protein expression of Eag1 channels in the human lung cancer cell lines A549 and NCI-H1975. Cell proliferation and survival were studied by the MTT method and the colony formation assay, respectively; apoptosis was investigated by flow cytometry. Gene expression was assessed by real-time polymerase chain reaction (RT-PCR), and protein expression was studied by Western blot analysis and immunocytochemistry. We obtained the inhibitory concentrations 20 and 50 (IC20 and IC50, respectively) values for each drug from the cell proliferation experiments. Drug combination at their IC20 had a superior effect by reducing cell proliferation and survival in up to 80% and 100%, respectively. The drugs alone did not affect apoptosis of H1975 cells, but the drug combination at their IC20 increased apoptosis roughly four times in comparison to the effect of the drugs alone. Eag1 mRNA levels and protein expression were decreased by the drug combination in A549 cells, and astemizole induced subcellular localization changes of the channel protein in these cells. Our in vitro studies strongly suggest that the combination astemizole-gefitinib may be a novel and promising therapy for lung cancer patients.

Astemizole inhibits cell proliferation in human prostate tumorigenic cells expressing ether à-go-go-1 potassium channels.

Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether à-go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue®  assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells.  Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.

Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells.

Oncogenic ether à-go-go-1 (Eag1) potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF) regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively) were obtained from the American Type Culture Collection and cultured following the manufacturer's recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development.

Biomonitoring of metal in children living in a mine tailings zone in Southern Mexico: A pilot study.

Accumulation of metals in soil represents a health risk for individuals living near mining areas, especially for children who have a higher susceptibility to metal related diseases. The Taxco mining district in Southern Mexico was one of the largest Mexican metal producers of silver and gold, among other metals. The aim of this pilot study was to evaluate metal exposure on children aged 6-11 years living in and around the Taxco mine tailings zone. Lead in blood (PbB) was measured by graphite furnace atomic absorption spectrophotometry (AAS). Urine arsenic (AsU) was measured by hydride generation AAS, urinary Hg (HgU) by flow injection cold vapor atomic absorption, and urinary concentration of other metals such as chromium (Cr), nickel (Ni), cadmium (Cd), barium (Ba), cobalt (Co), copper (Cu), zinc (Zn), manganese (Mn), molybdenum (Mo), strontium (Sr), and iron (Fe) were determined by inductively coupled plasma optical emission spectrometry. Fifty samples were analyzed for PbB, AsU, and HgU, and 35 samples for the other metals. The mean concentration+/-SD for each metal was: PbB, 9.4+/-3.3 microg/dL; NiU, 75.4+/-30.7 microg/L; BaU, 18.4+/-4.1 microg/L; MnU, 5.2+/-0.7 microg/L; CuU, 29.6+/-6.8 microg/L; AsU, 16.5+/-8.3 microg/L; HgU, 0.7+/-0.86 microg/L; CdU, 4.7+/-2.7 microg/L; CrU, 15.1+/-4.45 microg/L; CoU, 18.3+/-9.7 microg/L; SrU, 49.2+/-30.7 microg/L; ZnU, 628.4+/-438.9 microg/L; FeU, 30.5+/-17.7 microg/L; and MoU, 52.1+/-29.3 microg/L. Results of this exploratory study show that children residing in the mining area of Taxco were environmentally exposed to several metals and a high percentage of these children had levels of Ni, Ba, Mn, Cr, Co, Cd, As, Hg, and Pb above reference values. Thus, further studies are needed to assess the effects of simultaneous exposure to toxic metals in children residing in mining areas.

Arsenic alters monocyte superoxide anion and nitric oxide production in environmentally exposed children.

Arsenic (As) exposure has been associated with alterations in the immune system, studies in experimental models and adults have shown that these effects involve macrophage function; however, limited information is available on what type of effects could be induced in children. The aim of this study was to evaluate effects of As exposure, through the association of inorganic As (iAs) and its metabolites [monomethylated arsenic (MMA) and dimethylated arsenic (DMA)] with basal levels of nitric oxide (NO(-)) and superoxide anion (O(2)(-)), in peripheral blood mononuclear cells (PBMC) and monocytes, and NO(-) and O(2)(-) produced by activated monocytes. Hence, a cross-sectional study was conducted in 87 children (6-10 years old) who had been environmentally exposed to As through drinking water. Levels of urinary As species (iAs, MMA and DMA) were determined by hydride generation atomic absorption spectrometry, total As (tAs) represents the sum of iAs and its species; tAs urine levels ranged from 12.3 to 1411 microg/g creatinine. Using multiple linear regression models, iAs presented a positive and statistical association with basal NO(-) in PBMC (beta=0.0048, p=0.049) and monocytes (beta=0.0044, p=0.044), while basal O(2)(-) had a significant positive association with DMA (beta=0.0025, p=0.046). In activated monocytes, O(2)(-) showed a statistical and positive association with iAs (beta=0.0108, p=0.023), MMA (beta=0.0066, p=0.022), DMA (beta=0.0018, p=0.015), and tAs (beta=0.0013, p=0.015). We conclude that As exposure in the studied children was positively associated with basal levels of NO(-) and O(2)(-) in PBMC and monocytes, suggesting that As induces oxidative stress in circulating blood cells. Additionally, this study showed a positive association of O(2)(-) production with iAs and its metabolites in stimulated monocytes, supporting previous data that suggests that these cells, and particularly the O(2)(-) activation pathway, are relevant targets for As toxicity.

Estrogens and human papilloma virus oncogenes regulate human ether-à-go-go-1 potassium channel expression.

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation.

Genotoxic effects of environmental exposure to arsenic and lead on children in region Lagunera, Mexico.

DNA damage and DNA repair ability by means of the comet assay and the hydrogen peroxide challenge in lymphocytes from 65 children exposed simultaneously to As and Pb in Region Lagunera, Mexico. The first exposure scenario was concerned with natural As contamination in drinking water affecting all children, particularly those attending the schools farthest from (Gomez Palacio) and closest to the smelter (Pedro Garcia). The second scenario related to additional Pb and As soil and dust contamination in the schools located in the smelter vicinity (Heroe de Nacozari and Pedro Garcia). Most children (93%) had As in urine (AsU) above 50 microg/L and 65% had blood Pb (PbB) above 10 microg/dL. The highest AsU median levels were observed in the school farthest from the smelter, whereas the highest PbB values were observed in the closest school. DNA damage and a decreased repair ability observed in children attending the schools were more severe than those reported for healthy Mexican children. However, the multivariate analysis did not show significant associations between DNA basal damage and PbB or AsU. Lymphocytes from 58% of the children did not respond to the peroxide challenge, and those had a more severe basal DNA damage. DNA repair capacity showed a slowed response and was negatively associated with AsU. Thus, in addition to reduced exposure, further studies are needed to ascertain if the deficiency in DNA repair is transient or if children are already displaying a mutator phenotype and are at risk of developing cancer.