RESUMO
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.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Proteína do Retinoblastoma/metabolismo , Retinoblastoma/genética , Astemizol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pré-Escolar , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Lactente , Masculino , Oncogenes , RNA Mensageiro , Neoplasias da Retina/genética , Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genética , TransfecçãoRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Astemizol/farmacologia , Proliferação de Células/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Human ether à-go-go 1 (Eag1) potassium channels are potential tumor markers and therapeutic targets for several types of malignancies, including cervical cancer. Estrogens and human papilloma virus oncogenes regulate Eag1 gene expression, suggesting that Eag1 may already be present in pre-malignant lesions. Therefore, Eag1 could be used as an early marker and/or a potential risk indicator for cervical cancer. Consequently, we studied Eag1 protein expression by immunochemistry in cervical cancer cell lines, normal keratinocytes, cervical cytologies from intraepithelial lesions, biopsies from cervical intraepithelial neoplasias (CIN 1, 2 and 3) and in normal smears from patients taking or not taking estrogens. Two hundred and eighty-six samples obtained by liquid-based cytology and fifteen CIN biopsies were studied. We observed Eag1 protein expression in the cervical cancer cell lines, as opposed to normal keratinocytes. Eag1 was found in 67% of the cervical cytologies from low-grade intra-epithelial lesions and in 92% of the samples from high-grade intraepithelial lesions, but only in 27% of the normal samples. Noteworthy, morphologically normal cells obtained from dysplastic samples also exhibited Eag1 expression. In CIN biopsies we found that the higher the grade of the lesion, the broader the Eag1 protein distribution. Almost 50% of the normal patients taking estrogens displayed Eag1 expression. We suggest Eag1 as a potential marker of cervical dysplasia and a risk indicator for developing cervical lesions in patients taking estrogens. Eag1 detection in cervical cancer screening programs should help to improve early diagnosis and decrease mortality rates from this disease.