| PurposeCervical cancer is one of the mostly common gynecologic malignant tumors, and radiotherapy is the major therapy for patients of intermediate and advance cervical squamous cell carcinoma. Despite the new therapies like neutron irradiation and endovascular treatment between tissues, there is still a failure rate of 30%-40% mainly because of local recurrence and distant metastasis.The major casue of the failure of radiotherapy lies in that hypoxic cells in the central zone of tumor are insensitive to irradiation. Regular radiation dose is unable to eliminate all tumor cells. The rest tumor cells will recur arising out of rapid proliferation under the common involvement of multiple genes, among which, hypoxia inducible factor lα(HIF-1α) plays a leading role. HIF-1αis a nucleolus transcription factor most active in hypoxic state and expressed at high level in multiple tumor issues. HIF-1αis adjustable by many factors, and one of the major factors is hypoxia, of which the mechanism has not been identified yet.Eag1 potassium channel is a voltage-gated single channel consisting of 6 transmembrane domains (S1-S6) and being activated by depolarization. In normal case, Eag 1 potassium channel is distributed in a limited scope, mainly in the brain, and expressed at low level in myoblasts (before cell fusion) and placenta, and at high level in multiple tumors. At present, researchers hypothesize that each monomer of the tetramer formed by Eag channel contains 6 transmembranes with high content of voltage transducing proteins (positive-charge amino acids), and that at the C end in the cell is a Per-Arnt-Sim (PAS) district, i.e oxygen sensor, having high sensitivity to hypoxia. The reduction of von Hippel-Lindau gene (VHL) expression in tumor tissues will activate Eag1 channel and strengthen the stability of HIF-1α. The results indicate that Eag1 channel may plays an important role in the sensitivity of tumor cells to hypoxia and the adjustment mechanism of HIF-1α. By now, there is no report on the influence of Eag1 channel on the adjustment of HIF-1αof cervical cancer cells in hypoxic environment, The present study will serve as theoretical references for improvement of the effect of neutron-ray irradiation for intermediate and advance cervical cancer patients by analyzing the Eag1 expression and its activity change in hypoxic environment, the adjustment of Eag 1 on HIF-1α, and the influence of potassium channel antagonist on neutron-ray irradiation on Hela cell line.Methods1. Eag 1 expression and HIF-1αexpression in different cervical tissues were measured by immunohistochemistry. The characteristics of Eag 1 and HIF-1αexpression in Hela cell line in different hypoxic conditions or in the environment by using antagonist, and the relevance of such expressions with hypoxic conditions or in the antagonist environement were analyzed by immunofluorescence, RT-PCR and Western-blot methods.2. The differences of the electrophysiological characteristics of Eag 1channel on the hela cell line in normoxic and hypoxic state was recorded by using whole-cell patch clamps and single channel patch clamps to analyze the influence of hypoxia on Eag 1 channel activity in hypoxic state.3. The influence of Eag1 channel activity on Ca2+ concentration in cells, mitochondrial permeability transition pore (mPTP) and mitochondrial membrane potential (Δψ) in hypoxic state was measured by laser confocal method. The change of mitochondrial ultrastructure in hypoxic state was scanned with electron microscope. The influence of the open state of Eag 1 channel on concentrations of reactive oxygen species (ROS), ATP, ADP, and AMP and the energy generation in hypoxic state was measured by flow cytometry and high performance liquid chromatographic analysis.4. The influence of 4-aminopyridines (4-AP, the potassium antagonist) with different concentration on the inhibition rate of cell proliferation, apoptosis rate, the expressions of HIF-1αmRNA and proteins after neutron-ray irradiation on Hela cell line was measured by MTT, flow cytometry, RT-PCT and Western-blot methods. Results1. Through immunohistochemistry, Eag 1 expression and HIF-1αprotein expression in the cervical intraepithelial neoplasia tissues in the experimental group was significantly higher than that in the control group; Eag 1 expression and HIF-1αprotein expression in the cervical cancer tissues was significantly higher than that in the cervical intraepithelial neoplasia tissues; Eag 1 expression and HIF-1αprotein expression at I + II stage was significantly higher than that at Zero stage; and Eag 1 expression and HIF-1αprotein expression at III + IV stage was significantly higher than that at I + II stage. Eag1 expression and HIF-1αwere almost irrelevant to pathological grade, but those two were positively correlated to each other (r=0.520,p<0.01).Through immunofluorescence, RT-PCT and Western-blot, Eag 1 expression and HIF-1αexpression were found in Hela cell line of cervical cancer patients in normal culture condition and ascended in hypoxic state as increase of hypoxia and extension of the hypoxic time. Eagl channel antagonist can cut down HIF-1αexpression and slow down its translocation significantly.2. Through the records from whole-cell patch clamps, Eag 1 channel in healthy Hela cell line was voltage-gated, had delayed recifier and would not lose activity. Mild hypoxia could increase the membrane potential of Eag 1 channel, the current intensity and reduce the activation time.3. The influence of Eag 1 channel on the intracellular environment of hela cell line of cervical cancer patient in mild hypoxia includes: 1) rise of Ca2+ concentration, 2) open mPTP, 3)Δψreduction, 4) increase of mitochondria quantity and volume, and increase of mitochondrial cristae; 4) more ROS generated, 5) respiratory function of mitochondria strengthened, and 6) increase of cell storage capacity. However, Eag 1 channel antagonist could slow down the above changes.4. By adding 4-APs with different concentrations, the inhibition rate of Hela cell proliferation, and apoptosis rate were positively correlated to 4-AP concentration, and HIF-1αMrna expression and protein expression were negatively correlated to 4-AP concentration in the same hypoxic state with the equivalent neutron irradiation dose.Conclusions1. Both Eag 1 expression and HIF-1αexpression are related to the development of cervical cancer, but almost irrelevant to pathological grade. In detail, Eag 1 expression and HIF-1αexpression ascend as the development of clinical stage of cervical cancer, and Eag 1 expression is positively correlated to HIF-1αexpression. Eag1mRNA expression in Hela cell line cultured in normal environment is influenced by the severity of and endurance time to hypoxia. In hypoxic state, Eag 1 channel can adjust HIF-1Αexpression to some extent.2. The change of electrophysiological activities of Eag1 channel on the membrane of Hela cell in hypoxic environment indicates the significance of Eag1 in hypoxia sensing.3. The change of Eag 1 channel activity is critical to mitochondrial structure and function. Eag 1 may adjust HIF-1αexpression by changing mitochondrial structure and function in hypoxic environment.4. 4-AP can remarkably cut down the expression level of HIF-1αmRNA expression and protein expression in Hela cell line, increase the inhibition rate and apoptosis rate of cell proliferation and improve the therapeutic effect under the same hypoxic conditions with the equivalent neutron irradiation dose, which sheds some light on new research on radiotherapy for cervical cancer.
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