Research On Ion Channel Characteristics And Mechanisms Of Glioma Inhibition By Tamoxifen In SHG-44 Glioma Cell Line

Object: Glioma originates from glial cells,it remains the most common primary neoplasm of the central nervous system and accounts for approximately 50～60% of all brain tumors. Although many improvements have been made,current therapeutic modalityies including surgical resections,chemotherapy,radiotherapy or combinations cann't ensure a cure,especially to the high-grade gliomas which are insensitive to chemotherapy or radiotherapy.The treatment of gliomas has become one of the most difficult problems in neurosurgery.Hence,identification and characterization of the regulatory molecules that involved in the gliomas tumorigenesis may offer important targets for treatment strategies.Cellular membrane ion channels include sodiam channels,potassium channels,chloride channels and calcium channels.The opening of ion channels can cause changes of ions in cells.These changes will induce various kinds of physiological or pathological functions.Each channel can be divided into many subtypes which have different gating and regulatory modes.The same chnnel in different tissues may differ in distribution density,channel characteristics,regulatory styles or physiological functions.Ion channels play important roles in glioma cells.Potassium channels are related to cell proliferation and invasion whereas sodium channels are related to cell division. The cytomorphosis mediated by chloride channels is very important to glioma invation.Because of the important effects of ion channels in glioma proliferation,they may be taken as molecular targets of glioma therapeutics.To study the charateristics and regulations of ion channels in glioma cell membrane is helpful to reveal the pathophysiological mechanisms of gliomas and will provide important theoretical basis for new therapeutics. Some researches on glioma cell lines about ion channes and pharmacological regulations of cell multiplication and differentiation have been reported. Studies suggested that tamoxifen was beneficial in the treatment of brain tumors which was predominantly known as a competitive antagonist at the estrogen receptor and was used in the treatment of breast cancer. Studies in vitro proved tamoxifen could inhibit glioma cell's multiplication and migration. However, its complete mechanisms of action were more complex and not clearly delineated.Recent researches found tamoxifen could regulate ion chnnels,which may be related to its effect of tumor inhibition.But its regulatory mechanisms were unclear and needed to be clarified.C-fos gene is a important member of immediate early genes.The proteins coded by c-fos are important transcription factors in eukaryotic cells and can induce the mRNA transcription and protein expression of downstream reporter genes,which participate in the regulation of cell proliferation and apoptosis.Its unusual increase of expression will break the balance of cell multiplication and apoptosis,which may cause cell transformation and tumor generation.The regulation of c-fos may alter the tumor cell proliferation characteristics.It has complicated regulation styles in which PKC play important roles.To reveal its regulatory mechanisms is very helpful to tumor prevention.Glioma cell line SHG-44 has stable biological characteristics and is easy to culture,so it is generally applied in medical researches.The researches of its gene regulations,ion channels characteristics and regulations were scarcely reported.Respecting the importance of ion channels,c-fos gene expression and regulation to the growth,proliferation,invasion and apoptosis of SHG-44 cells,we carryed out this research.Our research consisted of three parts.Part I: The Inhibition of Proliferation on SHG-44 Cells by Tamoxifen Methods:1.SHG-44 cells were revitalized and cultured.To observe the cells growth conditions and changes in cell forms under microscope after various concentrations of tamoxifen were applied.2. To observe the expression of PKCαand ER through immunohistochemical techniques.Outcomes judgement:Buffy particles emerged in cytoplasm meaned PKC-positive whereas in cell nucleus meaned ER-positive.3.To observe the changes of survival rate after 0 to 15μmol/L tamoxifen was applied to the SHG-44 cells for one to three days by means of MTT test.4.To observe the changes of apoptosis rate and cell cycles of SHG-44 cells after 0,2,4,6μmol/L tamoxifen was applied by means of flow cytometry.5.Data were analyzed by use of SPSS 12.0 software. Results were expressed as mean±SD, and n represented the number of the cells examined. The results were considered to be significant when the P value was less than 0.05.Results:1.SHG-44 cells grew well in DMEM medium.The cells had fusiform shape and nucleus heteromorphism was universal.The freezing and revitalization had no influence on the biological characteristics.After tamoxifen was applied ,the refraction weaked,contour enhanced,synapses decreased,a great quantity of particles emerged in cytoplasm.The gaps between cells augmented. Cells faded and growth inhibited.The control cells grew normally.2. Buffy particles emerged in cytoplasm after PKCαantibody was applied while no changes took place after ER antibody was applied,which meant PKC expressed in SHG-44 cells.3.MTT test results:Compared with control cells,the absorbtance values obviously decreased after different concentrations of tamoxifen were applied.When the tamoxifen concentrations rose from 2μmol/L to 10μmol/L,the inhibition rate also increased.When the tamoxifen concentration exceeded 10μmol/L,the changes of inhibition rate were not obvious.Under the same tamoxifen concentration,the inhibition rate of cells in 48h group was obviously higher than that in 24h group.But compared with the 48h group, in the 72h group,only the inhibition rate of SHG-44 cells in 6μmol/L tamoxifen group increased.4.Flow cytometry test results:Compared with the control group of cells,the proportion of SHG-44 cells in G0/G1 stage decreased while the proportion of SHG-44 cells in G2/M or S stages increased after tamoxifen was applied.Cell apoptosis detection results:Compared with the control group,the proportion of apoptotic cells increased gradually when the concentration of tamoxifen in substratum increased.Part II The Charactersitics and regulation by tamoxifen of Ion Channels in SHG-44 CellsMethods:The SHG-44 cells were cultured.They were digested by 0.25% trypsin and 0.02% EDTA and made cell suspension and inoculated in the serum-free medium in which coverslips were placed in advance.After 48 hours,the whole-cell patch clamp technique was adopted to record the sodium channels,inward-rectified potassium channels and chloride channels in the cell membrane of SHG-44 cells.The channel currents would be recorded in 5 minutes after the rupture of membrane.2.To plot the I-V curves of each channel before and after different concentrations of tamoxifen were added to the extracellular fluid and observe the changes of summit current densities.3.I/Imax were plotted as a function of tamoxifen concentrations and were well fitted with hill function by Origin 7.5 software.The half inhibition concentrations(IC50)of each channel were calculated.4.The channel conductance of control group and tamoxifen treatment groups were normalized and plotted as a function of membrane potentials and were well fitted with Boltzmann function.The half activation voltages (V1/2)and k values could be calculated from the activation curves.To observe the influence of tamoxifen on the activation curve of each channel.5.To compare the chloride channel currents of tamoxifen treatment group,PKC inhibitor treatment group,PKC activator treatment group,tamoxifen and PKC inhibitor treatment group and analyse if the inhibition of ion channel currents in SHG-44 cells was related to PKC activity and its extent.The concentration of tamoxifen was 1.5μmol/L,PKC inhibitor was 100nmol/L staurosporine,PKC activator was 1μmol/L PMA.6. Data were analyzed by use of SPSS 12.0 software. Results were expressed as mean±SD, and n represented the number of the cells examined. The results were considered to be significant when the P value was less than 0.05.Results:1.The sodium currents averaged -254±176pA and activated and inactivated quickly.The threshold activation voltage was about -30mV.It was completely blocked by 1μmol/L TTX.Its V1/2 was -29.3±3.5mV,k value was 3.56±0.41.At the holding voltage of 0mV,the inhibition ratios of 2μmol/L, 6μmol/L,8μmol/L tamoxifen were 27.3±3.1%, 57.1±5.8%, 70.9±8.2%. The IC50 was 5.54±0.46μmol/L. Tamoxifen didn't alter the activation kinetics of sodium channels.2. The KIR currents averaged -375±76pA and activated quickly and inactivated slowly and was inward rectified.Its V1/2 was -75.32±5.4mV and k value was 12.26±1.3.At the holding voltage of -140mV,the inhibition ratios of 2μmol/L,6μmol/L,8μmol/L tamoxifen were 38.7±4.1%, 69.4±6.1%, 78.7±7.2%. The IC50 was 2.84±0.31μmol/L. Tamoxifen shifted the activation curve to the right.The V1/2 was -54.95±4.8 mV and k value was 16.17±1.8 in the 8μmol/L tamoxifen.3. The chloride currents averaged -2030±1956pA and activated quickly and didn't inactivate and was outward rectified.Its V1/2 was 34.14±3.2mV and k value was 20.59±2.3.At the holding voltage of +100mV,the inhibition ratios of 2μmol/L and 8μmol/L tamoxifen were 67.1±7.1% and 89.7±8.8%.The IC50 was 1.34±0.22μmol/L.Tamoxifen shifted the activation curve to the right.The V1/2 was -54.95±4.8mV and k value was 16.17±1.8.4.At the holding voltage of +100mV, 1.5μmol/L tamoxifen inhibited the chloride currents 48±5.6%. 100nmol/L staurosporine inhibited the chloride currents 42±4.8%. 1.5μmol/L tamoxifen and 100nmol/L staurosporine inhibited the chloride currents 39±5.3%. 1.5μmol/L tamoxifen could inhibit the chloride currents in staurosporine treatment group 7.1%. 1μmol/L PMA increased the chloride currents 36±4.1%. Part III Influence of c-fos Gene Expreesion in SHG-44 cells by TamoxifenMethods: The SHG-44 cells were cultured in medium containing 0μmol/L,2μmol/L,6μmol/L and 8μmol/L tamoxifen for 2 days.Total RNA of each treatment group was extracted and its integrity was checked.RNA was inverse transcripted to cDNA. Fluorescent quantitative PCR technique was used to amplify and quantitate the c-fos gene andβ-actin gene. Ct compared method was used to judge the inhibition extent of c-fos gene expression by different concentrations of tamoxifen.Results:The Ct values of c-fos gene expression in treatment groups of 0μmol/L,2μmol/L,6μmol/L and 8μmol/L tamoxifen were 17.14 ,18.48,19.8 and 20.55,The Ct values ofβ-actin were 18.01,17.62,18.81 and 17.42 accordingly.The silence efficiencies of c-fos gene in treatment groups of 2μmol/L,6μmol/L and 8μmol/L were 0.7,0.72 and 0.94.Conclusions:1.Tamoxifen could inhibit the proliferation of SHG-44 cells in which PKC expressed but ER not.The inhibition was time and drug concentration dependent but had certain time and concentration extent which was related to the inhibition of PKC activity.2.Tamoxifen blocked the SHG-44 cells at G2/M and S stages and increased the proportion of apoptosis cells.The inhibion effect of tamoxifen on SHG-44 cells was different from that on other tumor cells.3.Sodium channels expressed in SHG-44 cells whose electrophysiological characteristics were different from that in other tumor cells.Tamoxifen inhibited its currents concentration-dependently but didn't alter its activation kinetics.Its IC50 was 5.54μmol/L. 4. Inward potassium channels expressed in SHG-44 cells whose currents were inward rectified.Tamoxifen inhibited its currents concentration-dependently and shifted its activation curve to the right and made it difficult to activate. Its IC50 was 2.84μmol/L.5. Chloride channels expressed in SHG-44 cells whose currents were outward rectified.Tamoxifen inhibited its currents concentration-dependently and shifted its activation curve to the right and made it difficult to activate. Its IC50 was 1.34μmol/L.Chloride currents was the most sensitive to tamoxifen in the three kinds of channels we studied.6.The inhition of ion channels in SHG-44 cells by tamoxifen was mainly because of its inhition of protein phosphorylation by PKC.The characteristics and regulations of ion channels in SHG-44 cells were significantly different from that in many other tumor cells.7.Tamoxifen inhibited c-fos gene expression concentration-dependently.The possible mechanism was the inhibition of PKC activity by tamoxifen.8.The inhibition of cell proliferation and promotion of cell apoptosis by tamoxifen in SHG-44 cells were related to the inhibiton of ion channels and c-fos gene expression in which PKC acted as an important mediator.