Cervical cancer is a major cause of cancer mortality among women, particularly in developing countries.1,2 Therefore, new therapeutic options are needed. Repositioning of old drugs with novel anticancer effects is an alternative approach to fight cancer. For example, thalidomide has gained great interest for its potential use in multiple myeloma treatment.3 Likewise, the antihistamine astemizole has gained interest as a novel anticancer drug.4–6 In addition to binding to histamine receptor 1, astemizole targets other proteins involved in cancer including the P-glycoprotein and the K+ channels Eag1 (ether à-go-go-1, KCNH1, Kv10.1) and Erg (Eag-related gene).4–12
Eag1 is a voltage-gated K+ channel that shows restricted expression in healthy tissues; it is mainly found in brain, but it is also slightly expressed in the placenta, testes, adrenal glands, and transiently in myoblasts.13–15 In contrast, Eag1 expression has been observed in most types of tumors including breast, prostate, colon, ovarian, and cervical cancers, as well as leukemia, melanoma, and neuroblastoma. Therefore, Eag1 has been suggested as tumor marker.5,13,15–20 Because of its oncogenic properties,13 Eag1 has been also proposed as a target for anticancer therapy. Inhibition of Eag1 expression (with antisense oligonucleotides or small interfering RNA) or channel activity (with nonspecific blockers like imipramine or astemizole or specifically with monoclonal antibodies) has been used as an alternative approach to reduce proliferation of tumor cells in vitro and/or in vivo.5,12,13,21–26 Nevertheless, despite Eag1 messenger RNA (mRNA) and channel protein being expressed in cervical carcinoma, the effect of astemizole in cervical cancer cells is unknown. Therefore, we wondered whether astemizole affects proliferation or apoptosis of cervical cells expressing Eag1 channels.
MATERIALS AND METHODS
Cell lines and Reagents
The human cancer cervical cell lines HeLa, SiHa, CaSki, INBL, and C-33A were obtained from the American Type Culture Collection (Manassas, Va) and cultured according to manufacturer’s instructions. Astemizole was kindly provided by Liomont Laboratories (Mexico City), and imipramine and DMSO were purchased from Sigma Chemical Co (St. Louis, Mo). Anti-Eag1 monoclonal antibody was purchased from Novus International (Littleton, Co).
Cell lines were grown on charged glass slides and boiled for antigen retrieval, then blocked with endogenous peroxidase blocker (Bio SB, Santa Barbara, Calif) for 10 minutes and then incubated in the presence of 1:500 anti-Eag1 monoclonal antibody (Novus International) overnight at −4°C. The slides were then incubated with secondary biotin-antibody (Bio SB) for 15 minutes and then incubated with streptavidin polymer (Bio SB) for 15 minutes. The specific staining reaction was completed by incubating the slides in the presence of diaminobenzidine in buffer reaction solution (Bio SB) and observed as a brown staining. Sections were counterstained with hematoxylin (Dako, Glostrup, Denmark). The slides were observed in an Olympus IX51 microscope, Olympus DP70 camera. Previously obtained human placenta tissue sections27 were used as positive control for Eag1 protein expression.21,27
Cell proliferation was studied as previously described.21 Briefly, cells were seeded in 96-well plates, and cell proliferation was assayed by the colorimetric method based on the conversion of the tetrazolium salts to formazan crystals by dehydrogenase activity in active mitochondria [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation kit I, Boehringer Mannheim, GmbH]. Cells were incubated during 96 hours in culture medium alone or in the presence of astemizole (2 and 5 μmol/L), imipramine (10 μmol/L), or vehicle (DMSO). MTT (0.5 mg/mL) was added 4 hours before completing the total incubation time. Absorbance values were obtained from the resulting colored solution with a microplate photometer (Sunrise Touchscreen).
Apoptosis was studied as described.21 Briefly, cells were seeded in culture plates and incubated during 96 hours in culture medium alone or in the presence of astemizole (2 and 5 μmol/L), imipramine (10 μmol/L), or vehicle (DMSO). Camptothecin (apoptosis inductor) and methanol (necrosis inductor) were used as positive controls and were added 24 hours before the end of the treatment. Apoptosis was determined with the Annexin V-FITC kit (Invitrogen Co) binding to phosphatidylserine and DNA staining by propidium iodide (PI). Experiments were performed with the flow cytometer CYAN ADP (Dako, Glostrup, Denmark). Percentages of viable (FITC- and PI-negative), apoptotic (FITC-positive and PI-negative), and late apoptotic (FITC- and PI-positive) cells were obtained by quadrant analysis using the Summit 4.3 software.
Analysis of variance followed by Tukey-Kramer test was used to compare data between different experimental groups. P < 0.05 was considered to be statistically significant. Analysis was made using the GraphPad Prism software version 5.0.
Eag1 Protein Expression in Cervical Cancer Cell Lines
We previously demonstrated Eag1 mRNA expression in the cell lines used in this study,21 as well as protein expression in HeLa and SiHa cells.27 Then, in this study, we first decided to investigate Eag1 protein expression in the cervical cancer cell lines CaSki, INBL, and C-33A. Strong Eag1 immunostaining was observed in the 3 cell lines studied (Fig. 1). Human placental tissue was used as positive control (Fig. 1D), as previously described.27 Eag1 immunostaining was not observed in CaSki cells in the absence of the primary antibody (Fig. 1E). Next, we wondered whether astemizole might have antiproliferative and proapoptotic effects in several cervical cancer cell lines.
Astemizole Decreases Cervical Cancer Cell Proliferation
Because Eag1 protein expression was observed in the cell lines, we studied the effect of 2 Eag1 nonspecific blockers such as imipramine and astemizole on the proliferation of 5 cervical cancer cell lines. Although imipramine almost did not affect cervical cancer cell proliferation, astemizole reduced cell proliferation in all the cell lines studied, especially at the 5-μM concentration (Fig. 2). In the case of CaSki cells, astemizole decreased cell proliferation in almost 40%.
Astemizole Induces Apoptosis in Cervical Cancer Cells
Figure 3A displays 3 representative flow cytometry plots showing significant populations of cells either viable (upper left plot), in late apoptosis (upper right plot), or in early apoptosis (lower left plot). In a similar manner to the antiproliferative effects, whereas imipramine did not influence cervical cancer cell apoptosis, astemizole (5 μM) significantly induced apoptosis in all the cell lines studied (Fig. 3B–F). In some cases, the percentage of apoptotic cells in the vehicle-treated group was only approximately 2% of the whole population. Noteworthy, astemizole increased apoptosis severalfold, and in some cases, more than 70% of the cell population became apoptotic after astemizole treatment. Taken together, these results suggest astemizole as a potential therapeutic agent for cervical cancer.
Cervical carcinoma is a major cause of cancer death among women, especially in developing countries.1,2 Thus, alternative therapeutic approaches are needed. Here, we show that astemizole decreases proliferation and induces apoptosis of cervical cancer cells expressing Eag1 channels.
We have proposed Eag1 as a cervical carcinoma and cervical dysplasia marker.16,21,27 Actually, we detected channel activity in cervical cancer primary cultures, suggesting Eag1 as a potential target for cervical cancer treatment.16 Because of its role in cell proliferation, Eag1 channels have gained great interest in oncology.5,13,15,28 Astemizole has been suggested as a novel anticancer drug4–6 and has proven antiproliferative effects in tumor cells expressing Eag1 channels, including breast cancer and melanoma cells.12,26 Nevertheless, despite Eag1 mRNA, protein expression, and channel activity in cervical cancer cells, the effect of astemizole on the proliferation and apoptosis of these cells remained unknown. Here, we show Eag1 channel protein in the cervical cancer cell lines CaSki, INBL, and C-33A, in which Eag1 mRNA expression has been reported.21 Then, we tested the effect of astemizole in 5 cervical cancer cell lines expressing Eag1 channels. Cell proliferation and apoptosis were significantly affected by astemizole in all the cell lines tested. Interestingly, imipramine—another Eag1 nonspecific blocker—affected only cell proliferation in a modest manner, suggesting that the effect of astemizole on cervical cancer cells might be mediated not only by blocking Eag1 channels. Astemizole targets the P-glycoprotein,8 which confers resistance to some cancer cells.29 Accordingly, astemizole reverses doxorubicin resistance in leukemia cells.8 Another target of astemizole is the human Erg potassium channel, which has been proposed as a selective advantage for cancer cells.9 Then, because antihistamine treatment affects tumor cell proliferation,6,11 the antiproliferative effect of astemizole might be the result of targeting several proteins involved in cancer including histamine receptor 1, the P-glycoprotein, and the potassium channels Eag1 and Erg.
Because Erg potassium channels participate in heart ventricular repolarization, Erg channel blockers have been associated to cardiac arrhythmias and the LQT-2 syndrome.10 Astemizole overdosing produced some cardiovascular adverse effects and was withdrawn from some markets.4,6 However, astemizole at 50 mg/kg inhibits tumor growth in mice without producing obvious adverse effects.22 The 50-mg/kg astemizole dose is far below from the 2052-mg/kg astemizole lethal dose indicated by the Drug Bank.6 Thus, a proper cardiac function follow-up during astemizole treatment (without overdosing) might reposition astemizole as a safe cervical cancer treatment.
Cervical cancer is a multifactorial disease, and the major risk factor for the development of the disease is human papillomavirus (HPV) infection.1 Interestingly, Eag1 expression is regulated by HPV oncogenes and p53.21,30 Astemizole decreases metabolic activity of normal keratinocytes stably transfected with the E6/E7 HPV oncogenes.21 In the present study, we observed antiproliferative and proapoptotic effects of astemizole in cervical cancer cell lines with different HPV status. CaSki and SiHa cells express HPV 16, while HeLa and INBL cells display HPV 18 expression, and C-33A cells lack HPV. Therefore, astemizole may be used for cervical cancer treatment regardless of the HPV status. Our results suggest astemizole as an alternative therapeutic option for cervical cancer treatment.
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