Eag1 Expression Research Articles

Immunohistochemistry staining of Eag1 and p16/Ki-67 can help improve the management of patients with cervical intraepithelial Neoplasia after cold knife conversion

BackgroundImmunohistochemistry (IHC) is widely used in the management of patients with cervical intraepithelial neoplasia (CIN) but still has many limitations in clinical practice. We analyzed the correlation of new biomarkers with the severity of CIN and follow-up outcomes in patients after conization to improve the management of patients with CIN.MethodsIHC staining of Eag1 and p16/Ki-67 was performed on cervical tissue sections from 234 patients with suspected CIN2/3. After a series of follow-ups, including human papillomavirus (HPV) test and thinprep cytologic test (TCT) for 1–2 years, the outcomes were collected. IHC scores of biomarkers and follow-up results were used to analyze the correlation and assess the diagnostic efficiency of biomarkers.ResultsThe IHC staining intensity of Eag1 and p16/Ki-67 was significantly different from that of the CIN1-3 groups (p < 0.05). Eag1 expression scores were significantly different in the distribution between the two follow-up groups (p < 0.001). ROC curves based on the correlations between the follow-up outcomes and the Eag1 scores and IS of p16/ki-67 showed that Eag1 had a greater AUC (0.767 vs. 0.666). Logistic regression analysis of the combination of biomarkers revealed a greater AUC value than any single biomarker.ConclusionsEag1 expression was significantly correlated with CIN grade and follow-up outcomes after conization. IHC staining of combinations of biomarkers of Eag1, p16 and Ki-67 may help us to improve the ability to identify risk groups with abnormal follow-up outcomes after treatment for CIN.

The mechanism study of Eag1 potassium channel in gastric cancer.

Heredity factors may play a vital role in gastric cancer (GC) progression. This study is aimed to explore and validate the influence and the role of Eag1 on the susceptibility to GC. The successfully constructed Ad5-Eag1-shRNA vector was transfected into GC cells [SGC-7901 and BGC-823, short hairpin RNA (shRNA) group]. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting were conducted for assessment of Eag1 messenger RNA (mRNA) and protein expression levels. Cell proliferation and cell colony formation was measured by Cell Counting Kit-8 (CCK-8) assays. Flow cytometry was performed for cell cycle progression assessment. Bioinformatic analysis was analyzed for Eag1 validation with multiple public databases. The expression of Eag1 was significantly down-regulated in the shRNA group in comparison with the empty vector and control groups (P<0.05). Cell proliferation rate and clone formation number were lower in the shRNA group, and a decreased cell proportion in G2-S phase and an increased proportion in G1-G0 were observed in the shRNA group (P<0.05). When transfected with Ad5-Eag1-shRNA, cyclin D1 and cyclin E protein expression were inhibited. Bioinformatic analysis showed that Eag1 expression was strongly associated with the prognosis and immune infiltration of GC. The Eag1 gene may affect occurrence and development of GC through regulating cyclin D1 and cyclin E expression.

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.

HANR promotes lymphangiogenesis of hepatocellular carcinoma via secreting miR-296 exosome and regulating EAG1/VEGFA signaling in HDLEC cells.

The long noncoding RNA HANR has been shown to be involved in the progression of hepatocellular carcinoma (HCC). However, the underlying mechanism of HCC-associated long noncoding RNA (HANR)-regulated HCC metastasis and lymphangiogenesis has not been elucidated. RT-qPCR and Western blot methods were utilized to detect the gene expressions. Interaction of HANR with miR-296 was predicted by a bioinformatic program and validated by a dual-luciferase reporter assay. For the functional experiment, a transwell invasion assay was utilized to examine the invasion abilities of HepG2 and Huh-7 cells. The lymphatic vessel formation assay was used to show the HCC-associated lymphatic vessel formation ability of human dermal lymphatic endothelial cells (HDLEC). HANR was shown to directly bind to miR-296, and miR-296 downregulated HANR expression in HepG2 cells. Then, we observed that miR-296 inhibitor transfection in shHANR HCC cells could promote lymphatic vessel formation and invasion of HDLEC cells compared with shHANR HCC cells. EAG1 or VEGFA overexpression in HDLEC cells rescued lymphatic vessel formation and invasion in HDLEC cells coincubated with the medium of HepG2 cells expressing shHANR or miR-296 mimic. Ultimately, HANR knockdown and miR-296 mimic led to a significant decrease in the EAG1 and VEGFA expression levels in HepG2 cells. Here, we reveal a novel molecular mechanism in which the HANR/miR-296/EAG1/VEGF axis is responsible for the lymphangiogenesis of HCC cells. Our findings provide more insights into developing therapeutical or diagnostic methods by targeting HANR.

MiRNA-34a inhibits human osteosarcoma proliferation by regulating the expression of ether-à-go-go 1 gene: a study in vitro

Objective To detect the influence of miRNA-34a (miR-34a) on the proliferation of osteosarcoma and the mechanisms responsible for miR-34a regulation. Methods The osteoblastic cell line MG-63 and Saos-2, human osteoblastic cell line hFOB 1.19, 10 osteosarcoma tissues and 10 normal bone tissues were selected. The expression of miRNA-34a in osteosarcoma cells and tissues was detected by quantitative real-time polymerase chain reaction (qPCR). Next, a eukaryotic expression vector named pcDNA/miR-34a was constructed. Then, osteosarcoma cells were transfected with this eukaryotic expression vector and the effects of miR-34a overexpression on the proliferation and growth of osteosarcoma were measured using CCK-8, colony formation and xenograft model of nude mice. Finally, Western blot analysis was used to detect the expression of ether-a-go-go 1 (Eag1) gene in osteosarcoma cells after transfected with pcDNA/miR-34a or a miR-34a inhibitor miR-34a-2'-O-Methyl antisense oligoribonucleotide (miR-34a-2'-O-Me). Results Compared with normal bone tissues and osteoblastic cell line, miR-34a was down-regulated in osteosarcoma cell lines and tissues. Compared with the blank group and the control group, the cell survival rates of miR-34a group of the two cell lines were significantly lower [MG-63 72 h: blank group (40.05±4.82)%, control group (36.88±4.66)%, miRNA-34a group (26.24±6.22)%; MG-63 96 h: blank group (83.55±5.95)%, control group (80.13±4.48)%, miRNA-34a group (30.21±7.26)%; Saos-2 72 h: blank group (46.45±8.15)%, control group (43.33±6.89)%, miRNA-34a group (26.81±3.17)%; Saos-2 96 h: blank group (84.79±4.10)%, control group (80.14±3.11)%, miRNA-34a group (31.77±5.17)%]. The similar results were obtained from colony formation assay (MG-63: blank group 83.40±3.29, control group 80.00±3.06, miR-34a group 24.40±2.71; Saos-2: blank group 85.00±3.32, control group 80.60±3.29, miR-34a group 30.40±4.94). The tumor volumes of osteosarcoma xenograft in the miR-34a group was significantly smaller than that in the blank group and control group after 21 days treatment (all P < 0.001). Overexpression of miR-34a could decrease Eag1 expression in osteosarcoma cell lines while inhibition of miR-34a induced the of expression Eag1(P < 0.001). Conclusion MiR-34a plays a tumor suppressor role in osteosarcoma and could suppress the proliferation and growth of osteosarcoma through the regulation of Eag1. Moreover, it may be a novel target for osteosarcoma therapy. Key words: Osteosarcoma; MicroRNAs; Cell proliferation; Mice, nude; Gene, ether-a-go-go 1

Expression of Ether à go-go 1 and its molecular mechanism of regulating the malignant phenotype of osteosarcoma

Objective: To explore the expression of ether à go-go 1 (Eag1) in human osteosarcoma and its molecular mechanisms of regulating the malignant phenotype of osteosarcoma. Methods: The expression levels of Eag1 in osteosarcoma cell lines and human osteosarcoma tissues were detected by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry. The small interfering RNA (siRNA) was used to inhibit the expression of Eag1. The abilities of proliferation and invasion in osteosarcoma cells transfected with Eag1 siRNA were determined by CCK-8, colony formation assay, transwell assay and wound healing assay. The osteosarcoma xenograft model of nude mouse was established and tumor growth curve was drawn. Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF) and signal transducer and activator of transcription 3 (STAT3) in osteosarcoma cells transfected with Eag1 siRNAs. Results: Eag1 was overexpressed in the osteosarcoma cells and tissues. Compared with the scrambled siRNA group, the cell survival rates of Eag1 siRNA1 and Eag1 siRNA2 groups of the two cell lines were significantly lower. [MG-63 cells: scrambled siRNA group (100.0±4.65)%, Eag1 siRNA1 group (63.57±3.89)%, and Eag1 siRNA2 group (54.13±3.70)%; Saos-2 cells: scrambled siRNA group (100.00±5.46)%, Eag1 siRNA1 group (56.70±5.34)%, and Eag1 siRNA2 group (40.27±5.28)% (P<0.001 for all)]. Similar results were obtained from colony formation assay. The colony formation rates of MG-63 cells: the scrambled siRNA group was (92.00±3.46)%, Eag1 siRNA1 group (60.00±3.06)%, and Eag1 siRNA2 group (53.67±2.40)%; the colony formation rates of Saos-2 cells: the scrambled siRNA group was (92.00±5.57)%, Eag1 siRNA1 group (52.33±5.13)%, and Eag1 siRNA2 group (41.67±2.73)%. Compared with the scrambled siRNA group, P<0.001 for all. The tumor volumes of osteosarcoma xenograft in the Eag1 siRNA1 and Eag1 siRNA2 groups were significantly smaller than that in the scrambled siRNA group after 10 days treatment (P<0.01 for all). The invasion assay data showed that MG-63 and Saos-2 cells transfected with Eag1 siRNAs exhibited the ability of cell invasion, when compared with the cells transfected with scrambled siRNA. (Invasive cell number of MG-63 cells: the scrambled siRNA group was 134.00±3.61, Eag1 siRNA1 group 105.20±2.52, and Eag1 siRNA2 group 91.00±3.01; Invasive cell number of Saos-2 cells: the scrambled siRNA group was 132.30±3.23, Eag1 siRNA1 group 114.30±3.48, and Eag1 siRNA2 group 82.67±6.33. Compared with the scrambled siRNA group, P<0.01 for all. The migration rates were (62.48±1.83)%, (35.98±1.23)% and (32.30±1.20)% in the three groups of MG-63 cells, and (70.15±1.42)%, (41.38±1.34)% and (32.40±1.92)% in the three groups of Saos-2 cells, respectively. Compared with the scrambled siRNA group, P<0.001 for all. Notably, the expression levels of VEGF decreased evidently after Eag1 siRNAs transfection, paralleled with reduction in the expression levels of STAT3. Conclusions: Eag1 may promote osteosarcoma cell proliferation and invasion by targeting STAT3-VEGF pathway and may be a potential therapeutic target for osteosarcoma.

Knockdown of Eag1 Expression by RNA Interference Increases Chemosensitivity to Cisplatin in Ovarian Cancer Cells

Ether á go-go 1 (Eag1) is frequently highly expressed in various malignant cancers and its excessive expression is correlated with poor prognosis in various cancers. However, the relationship of Eag1 expression with the clinical outcome of patients having ovarian cancer treated with cisplatin-based adjuvant chemotherapy is still unknown. In this study, we measured the expression of Eag1 in ovarian cancer and investigated the association between cisplatin chemosensitivity of ovarian cancer cells and Eag1 expression level. We demonstrate that decreased expression of Eag1 correlates with favorable prognosis in patients treated with cisplatin-based adjuvant chemotherapy and predicts higher cisplatin sensitivity in ovarian cancer cells. In vitro, knockdown of Eag1 by small interfering RNA facilitated the sensitivity of ovarian cancer cells (SKOV3 and TYK) to cisplatin-induced apoptosis via nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) pathway. Furthermore, knockdown of Eag1 expression was associated with decreased expression of the P-glycoprotein without affecting multidrug resistance-associated protein 1 expression. Taken together, Eag1 may serve as a potential indicator to predict Eag1 chemosensitivity, and silencing Eag1 may represent a potential therapeutic strategy for ovarian cancer.

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.

In vivo dual targeting of the oncogenic Ether-à-go-go-1 potassium channel by calcitriol and astemizole results in enhanced antineoplastic effects in breast tumors.

BackgroundThe oncogenic ether-à-go-go-1 potassium channel (EAG1) activity and expression are necessary for cell cycle progression and tumorigenesis. The active vitamin D metabolite, calcitriol, and astemizole, a promising antineoplastic drug, target EAG1 by inhibiting its expression and blocking ion currents, respectively. We have previously shown a synergistic antiproliferative effect of calcitriol and astemizole in breast cancer cells in vitro, but the effect of this dual therapy in vivo has not been studied.MethodsIn the present study, we explored the combined antineoplastic effect of both drugs in vivo using mice xenografted with the human breast cancer cell line T-47D and a primary breast cancer-derived cell culture (MBCDF). Tumor-bearing athymic female mice were treated with oral astemizole (50 mg/kg/day) and/or intraperitoneal injections of calcitriol (0.03 μg/g body weight twice a week) during 3 weeks. Tumor sizes were measured thrice weekly. For mechanistic insights, we studied EAG1 expression by qPCR and Western blot. The expression of Ki-67 and the relative tumor volume were used as indicators of therapeutic efficacy.ResultsCompared to untreated controls, astemizole and calcitriol significantly reduced, while the coadministration of both drugs further suppressed, tumor growth (P < 0.05). In addition, the combined therapy significantly downregulated tumoral EAG1 and Ki-67 expression.ConclusionsThe concomitant administration of calcitriol and astemizole inhibited tumor growth more efficiently than each drug alone, which may be explained by the blocking of EAG1. These results provide the bases for further studies aimed at testing EAG1-dual targeting in breast cancer tumors expressing both EAG1 and the vitamin D receptor.

Positive correlation between the expression of hEag1 and HIF-1α in breast cancers: an observational study

ObjectivesTo explore the expression patterns of Eag1 (ether á go-go 1) and HIF-1α (hypoxia-inducible factor 1α) in a cohort of patients with breast cancer.SettingDepartment of general surgery in an upper...

Silencing of Ether à Go-Go 1 by shRNA Inhibits Osteosarcoma Growth and Cell Cycle Progression

Recently, a member of the voltage-dependent potassium channel (Kv) family, the Ether à go-go 1 (Eag1) channel was found to be necessary for cell proliferation, cycle progression and tumorigenesis. However, the therapeutic potential of the Eag1 channel in osteosarcoma remains elusive. In the present study, a recombinant adenovirus harboring shRNA against Eag1 was constructed to silence Eag1 expression in human osteosarcoma MG-63 cells. We observed that Eag1-shRNA inhibited the proliferation and colony formation of MG-63 cells due to the induction of G1 phase arrest. Moreover, in vivo experiments showed that Eag1-shRNA inhibited osteosarcoma growth in a xenograft nude mice model. In addition, selective inhibition of Eag1 significantly decreased the expression levels of cyclin D1 and E. Taken together, our data suggest that the Eag1 channel plays a crucial role in regulating the proliferation and cell cycle of osteosarcoma cells, and represents a new and effective therapeutic target for osteosarcoma.

P02.06 - Regulation of Ether À-Go-Go-1 Potassium Channel (EAG1) Gene Expression by Serum and TGF-Alpha in Human Liver Cancer Cells

Eag1 display oncogenic properties, and are proposed as markers and therapeutic cancer targets (1 - 5). Nevertheless, Eag1 regulation by endogenous factors is poorly understood. Because of the restricted distribution of Eag1 in normal tissues, we wondered whether serum regulates Eag1 expression. We also looked for ERK/MAPK activation and studied the effect of TGF-alpha (which activates ERK/MAPK) on Eag1 gene expression.

MicroRNA-34a Inhibits Human Osteosarcoma Proliferation by Downregulating Ether à go-go 1 Expression

Aberrant expression of MicroRNAs (miRNAs) has been implicated in several types of cancer. As a direct target gene of p53, miR-34a has been suggested to mediate the tumor suppressor function of p53. Ether à go-go 1 (Eag1) channel is overexpressed in a variety of cancers and plays important roles in cancer progression. However, the link between miR-34a and Eag1 in cancer is unclear. In this study, we used human osteosarcoma as the model to demonstrate that miR-34a was significantly downregulated in osteosarcoma tissues and cell lines compared with normal brain tissues and osteoblastic cell line. Next we evaluated the role of miR-34a in the regulation of osteosarcoma cell proliferation by CCK-8 and colony formation assays. The results showed that overexpression of miR-34a inhibited the proliferation of MG-63 and Saos-2 cells. Furthermore, xenograft nude mice model showed that miR-34a inhibited osteosarcoma growth in vivo. Mechanistically, we found that overexpression of miR-34a led to decreased Eag1 expression in osteosarcoma cells while inhibition of miR-34a increased Eag1 expression. Taken together, our results suggest that miR-34a could inhibit osteosarcoma growth via the down regulation of Eag1 expression.

K+ channels and cell cycle progression in tumor cells

K+ ions play a major role in many cellular processes. The deregulation of K+ signaling is associated with a variety of diseases such as hypertension, atherosclerosis, or diabetes. K+ ions are important for setting the membrane potential, the driving force for Ca2+ influx, and regulate volume of growing cells. Moreover, it is increasingly recognized that K+ channels control cell proliferation through a novel signaling mechanisms triggered and modulated independently of ion fluxes. In cancer, aberrant expression, regulation and/or sublocalization of K+ channels can alter the downstream signals that converge on the cell cycle machinery. Various K+ channels are involved in cell cycle progression and are needed only at particular stages of the cell cycle. Consistent with this idea, the expression of Eag1 and HERG channels fluctuate along the cell cycle. Despite of acquired knowledge, our understanding of K+ channels functioning in cancer cells requires further studies. These include identifying the molecular mechanisms controlling the cell cycle machinery. By understanding how K+ channels regulate cell cycle progression in cancer cells, we will gain insights into how cancer cells subvert the need for K+ signal and its downstream targets to proliferate.

Abstract P6-04-29: Vitamin D induces expression of estrogen receptor and restores endocrine therapy response in estrogen receptor-negative breast cancer

Abstract Background: Approximately 30% of all breast tumors do not express estrogen receptor (ER) and patients with these tumors present poor prognosis and respond poorly to hormone therapy. Calcitriol through its vitamin D receptor (VDR) exerts antiproliferative, apoptotic and pro-differentiating effects in cancer. Calcitriols effects upon ER expression in breast cancer cells is controversial. Therefore, in order to clarify this issue, the aim of the present study was to determine if calcitriol induces ERα expression in ERα-negative breast cancer cells and could restore antiestrogen responses. The evaluation of calcitriol effects was performed in terms of proliferation and regulation of the following genes: Cyclin D1, involved in cell cycle, and Ether-à-go-go 1 (Eag1), related to cell proliferation and tumor progression. Methods: Cultured cells derived from ERα-negative breast tumors and an established ERα-negative breast cancer cell line (SUM 229) were used in this study. These cells were treated with calcitriol and reverse transcription-PCR or western blotting analyses were performed to assess ERα expression. Growth assays with XTT were used to evaluate the antiproliferative response to the antiestrogens fulvestran and tamoxifen. Gene expression analysis for Cyclin D1 and Eag1 was evaluated by real time PCR in cells treated simultaneously with calcitriol plus estradiol or fulvestran. Results: The treatment with calcitriol in ER-negative breast cancer cells resulted in the induction of ERα. This effect was specifically mediated through the vitamin D3 receptor (VDR), since the VDR antagonist TEI-9647 effectively inhibited the ability of calcitriol to stimulate ERα gene expression. Consequently, the induction of ERα by calcitriol restores the response to antiestrogens in breast cancer cells by inhibiting cell proliferation. Co-treatment of calcitriol and antiestrogens down-regulated Cyclin D1 and Eag1 gene expression. Conclusion: Calcitriol induced the expression of ERα and restored antiestrogenic responses in ERα-negative breast cancer cells. Moreover, fulvestran down regulated mRNA expression of Cyclin D1 and Eag1 when ERα-negative cells were pre-treated with calcitriol. These results suggest that the combined treatment with calcitriol and antiestrogens could be a new therapeutic strategy for ERα-negative breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-29.

Short Hairpin RNA (shRNA) Ether à go-go 1 (Eag1) Inhibition of Human Osteosarcoma Angiogenesis via VEGF/PI3K/AKT Signaling

Ether à go-go 1 (Eag1) channel is overexpressed in a variety of cancers but the therapeutic potential of Eag1 in osteosarcoma remains elusive. In this study, we constructed an Ad5-Eag1-shRNA vector and evaluated its efficiency for Eag1 knockdown and its effects on osteosarcoma. Our results showed that Ad5-Eag1-shRNA had high interference efficiency of Eag1 expression and suppressed osteosarcoma growth both in vitro and in vivo. To explore the molecular mechanism underlying tumor growth inhibition induced by Eag1 silencing, the intratumoral microvessel density (MVD) was assessed by CD31 staining and the expression of vascular endothelial growth factor (VEGF) was detected by Western blot analysis. We found that Eag1 silencing led to decreased angiogenesis and VEGF expression in the xenograft model of osteosarcoma. Finally, we detected a time-dependent decrease in VEGF expression and considerably reduced phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) activation in osteosarcoma cells treated by Eag1 shRNA. Taken together, our results suggest that Eag1 silencing inhibits tumor growth and angiogenesis in osteosarcoma via the down regulation of VEGF/PI3K/AKT signaling.

MiR-296-3p regulates cell growth and multi-drug resistance of human glioblastoma by targeting ether-à-go-go (EAG1)

MicroRNAs (miRNAs) – short non-coding RNA molecules – post-transcriptionally regulate gene expressions and play crucial roles in diverse biological processes such as development, differentiation, apoptosis and proliferation. In order to investigate the possible role of miRNAs in the development of multi-drug resistance (MDR) in human glioblastoma, we first detected (by Western blotting, real-time polymerase chain reaction [RT-PCR] and immunohistochemistry) the expression of miR-296-3p and ether-à-go-go (EAG1 or KCNH1) in U251 cells, U251/imatinib mesylate (U251AR cells) and clinical specimens. The results showed that miR-296-3p was down-regulated in U251AR cells, concurrent with the up-regulation of EAG1 protein, compared with the parental U251 cell line. In vitro drug sensitivity assay demonstrated that over-expression of miR-296-3p sensitised glioblastoma (GBM) cells to anticancer drugs, whereas down-expression using antisense oligonucleotides conferred MDR. Ectopic expression of miR-296-3p reduced EAG1 expression and suppressed cell proliferation drug resistance, and the luciferase activity of an EAG1 3′-untranslated region-based reporter construct in U251AR cells, whereas EAG1 over-expression rescued the suppressive effect of miR-296-3p in U251AR cells. We also found that EAG1 was widely over-expressed and inversely correlated with miR-296-3p in clinical specimens. Taken together, our findings suggest that miR-296-3p may play a role of MDR in glioblastoma at least in part by targeting EAG1.

Astemizole Synergizes Calcitriol Antiproliferative Activity by Inhibiting CYP24A1 and Upregulating VDR: A Novel Approach for Breast Cancer Therapy

BackgroundCalcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1) expression, which is necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. Herein, we characterized the interaction between calcitriol and astemizole as well as their conjoint antiproliferative action in SUM-229PE, T-47D and primary tumor-derived breast cancer cells.Methodology/Principal FindingsMolecular markers were studied by immunocytochemistry, Western blot and real time PCR. Inhibitory concentrations were determined by dose-response curves and metabolic activity assays. At clinically achievable drug concentrations, synergistic antiproliferative interaction was observed between calcitriol and astemizole, as calculated by combination index analysis (CI <1). Astemizole significantly enhanced calcitriol’s growth-inhibitory effects (3–11 folds, P<0.01). Mean IC20 values were 1.82±2.41 nM and 1.62±0.75 µM; for calcitriol (in estrogen receptor negative cells) and astemizole, respectively. Real time PCR showed that both drugs alone downregulated, while simultaneous treatment further reduced Ki-67 and Eag1 gene expression (P<0.05). Astemizole inhibited basal and calcitriol-induced CYP24A1 and CYP3A4 mRNA expression (cytochromes involved in calcitriol and astemizole degradation) in breast and hepatoma cancer cells, respectively, while upregulated vitamin D receptor (VDR) expression.Conclusions/SignificanceAstemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, upregulating VDR and targeting Eag1. This study provides insight into the molecular mechanisms involved in astemizole-calcitriol combined antineoplastic effect, offering scientific support to test both compounds in combination in further preclinical and clinical studies of neoplasms expressing VDR and Eag1. VDR-negative tumors might also be sensitized to calcitriol antineoplastic effects by the use of astemizole. Herein we suggest a novel combined adjuvant therapy for the management of VDR/Eag1-expressing breast cancer tumors. Since astemizole improves calcitriol bioavailability and activity, decreased calcitriol dosing is advised for conjoint administration.

Anti‐miR‐155 oligonucleotide enhances chemosensitivity of U251 cell to taxol by inducing apoptosis

The oncogene, microRNA-155, is significantly elevated in GBM (glioblastoma multiforme), regulating multiple genes associated with cancer cell proliferation, apoptosis and invasiveness. Thus, miR-155 can theoretically become a target for enhancement of the chemotherapy in cancer. Down-regulating miR-155 to enhance the effect of taxol has not been studied in human GBM. Human GBM U251 cells were treated with taxol and the miR-155 inhibitor alone or in combination. IC50 values were dramatically decreased in cells treated with miR-155 inhibitor combined with taxol, to a greater extent than those treated with taxol alone. Furthermore, the miR-155 inhibitor significantly enhanced apoptosis in U251 cells. The data suggest that miR-155 blockage increased the chemosensitivity to taxol in GBM cells, making combined treatment an effective therapeutic strategy for controlling the growth by inhibiting EAG1 expression.

Eag1 Channels as Potential Cancer Biomarkers

Cancer is a leading cause of death worldwide. New early tumor markers are needed to treat the disease at curable stages. In addition, new therapeutic targets are required to treat patients not responding to available treatments. Ion channels play major roles in health and disease, including cancer. Actually, several ion channels have been suggested as potential tumor markers and therapeutic targets for different types of malignancies. One of most studied ion channels in cancer is the voltage-gated potassium channel Eag1 (ether à go-go 1), which has a high potential to be used as a cancer biomarker. Eag1 is expressed in most human tumors, in contrast to its restricted distribution in healthy tissues. Several findings suggest Eag1 as a potential early marker for cervical, colon, and breast cancer. In addition, because Eag1 amplification/expression is associated with poor survival in leukemia, colon and ovarian cancer patients, it has also been proposed as a prognosis marker. Moreover, inhibition of either expression or activity of Eag1 leads to reduced proliferation of cancer cells, making Eag1 a potential anticancer target. Using Eag1 in cancer detection programs could help to reduce mortality from this disease.