In Vivo And Vitro Studies On Effects Of Ion Channel Blockers On Proliferation, Apoptosis And RNA-dependent Adenosine Deaminase 1 In Human Laryngeal Carcinoma Hep-2 Cell

The mechanism of the occurrence and development of laryngeal carcinoma is complex.It is not clear today.Cellular malignant change is closely related to cellular signal transmitting.Abnormal cell signal transmitting plays an important role in the occurrence and development of malignancy.Cellular signal transmitting is effected by many facts,such as protein metabolism and ion channels.Firstly,there is plenty of protein metabolism during cell signal transmitting.RNA editing is the phenomenon of nucleotide variation of post-transcriptional mRNA and tRNA.It is closely related to protein metabolism.RNA editing is a process that modifies gene coding mRNA.If RNA editing changes,genetic information carried by RNA will be changed,protein sequence,structure and function also will be changed.Enzyme which effects RNA editing is RNA-dependent adenosine deaminase(ADAR).Studies discovered that abnormity expression of ADAR1 was closely related to proliferation of carcinoma cell.Secondly,ion channels of cell membrane changes in the process of cell signal transmitting.One side,potassium(K) ion channels exists widely in eukaryote cell from protozoa to mammalian.Delayed rectifier potassium channel is one of voltage-gated potassium channels.Not only in excitable cell does it play an important decisive role in repolarization phase of action potential and maintenance of refractory period,but also it is closely related to proliferation of carcinoma cell.On the other side,Cl~- is the most common anion.It is necessary for the cell biosynthesis,signal transduction,and function of membrane normal receptors.Recently,some reports showed that cell C1C plays a critical role in cell proliferation and differentiation.Another side,calcium ion channels are important in the signal transmitting access.The changes of intracellular calcium ion concentration can cause tumor cell growth.Calcium channel blockers can inhibit the proliferation of carcinoma cell by controlling influx and release of calcium ion.Although it is clear that cellular ion transport proteins is an important part of carcinoma cell,there is no data from literature reports about ion channels in laryngeal carcinoma in China and abroad nowadays.Therefore,in this study,perforated patch-clamp whole-cell recording technique, MTT colorimetric assay,crystal violet method,flow cytometry,TUNEL Staining, Western blot and reverse transcription-polymerase chain reaction(RT-PCR) were used to study the characteristics of ion channels in Hep-2 cells,phosphorylated ERK1/2,AKT1 protein level and the expression of ADAR1 mRNA,the relationships among blocking ion channels,apoptosis and proliferation in Hep-2 cells,to study the possible mechanism of its occurring and developing.To study the effection of chloride ion channels' blocker on tumor in vivo,Hep-2 cells were used to establish laryngeal carcinoma nude mice xenograft modes.Different groups were treated with different concentration of NPPB.Our studies were divided into three subsections.First part The characteristics of ion channels in Hep-2 cells and the expression of ADAR1 mRNA in human laryngeal carcinomaMethods1.Potassium ion channels,chloride ion channels and calcium ion channels were measured by perforated patch-clamp whole-cell recording technique.2.The expression of ADAR1 mRNA in laryngeal carcinoma tissues,tumor-adjacent tissues and Hep-2 cells were detected by using RT-PCR.3.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.t test and ANOVA analysis of variance was employed.The significant level was P＜0.05.Results 1.Resting membrane potential of Hep-2 cell was(-29.8±1.9)mV.A sort of voltage-dependent outwardly rectifying transmembrane current was recorded.This current could be blocked by TEA.2.Single channel chloride current of Hep-2 cell was outwardly rectifying, voltage-dependent and volume-sensitive.The chloride current could be blocked by NPPB.3.When sodium current and potassium current were blocked,voltage of clamp was -90 mV;calcium current of Hep-2 cell membrane was recorded continuously.When depolarized by10 mV step,calcium reverse current was recorded in -60 mV.Calcium current of Hep-2 cell membrane was low-voltage activated calcium channels.The calcium current could be blocked by ND.4.ADAR1 mRNA expressed in laryngeal carcinoma and corresponding para-carcinoma tissues.There were not significant differences among different clinic stages,different classes and metastasis of lymph(P＞0.05).5.ADAR1 mRNA expressed in Hep-2 cell.The relative expression of ADAR1 mRNA was 2.852±0.735.There was significant differences between Hep-2 cell and control group(P＜0.05).Second part The effects of blocking ion channels on Hep-2 cell proliferation, apoptosis,phosphorylated ERK1/2,AKT1 protein levels and the expression of ADAR1 mRNAMethods1.MTT colorimetric assay and crystal violet was used to determine Hep-2 cells proliferation before and after blocking ion channels.2.TUNEL staining was used to determine Hep-2 cells apoptosis before and after blocking ion channels.3.Cell cycle,apoptosis of Hep-2 cells was determined by flow cytometry.4.The total and phosphorylated ERK1/2 and AKT1 protein levels of Hep-2 cells were detected by Western blot before and after blocking ion channels.5.The expressions of ADAR1 mRNA were examined by RT-PCR before and after ion channels blocked in Hep-2 cells. 6.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.The differences among the groups were compared with the ANOVA.The relationships among the inhibited Hep-2 cells proliferation,phosphorylated ERK1/2 and AKT1 protein levels and expressions of ADAR1 mRNA were studed by Spearman analysis.The significant level was P＜0.05.Results1.Compared with control group,the blocker of potassium ion channel(5,10,15, 20mM TEA) could significantly inhibit the proliferation of Hep-2 cell and induce the apoptosis dose-effect dependently,time-effect dependently.Blocking potassium ion channel could increase the cell ratio of G0/G1.2.The blocker of chloride ion channel(50,100,150μM NPPB) suppressed proliferation of Hep-2 cell concentration dependently.When Hep-2 cell was affected by NPPB for 12h,cell apoptosis was induced,which showed obvious time-effect relationship. After affected by NPPB,the cell ratio of G0/G1 increased,the cell ratio of S decreased, which showed obvious time-effect and dose-effect relationship.3.The blocker of calcium ion channel(5,10,50,100μM ND) suppressed proliferation of Hep-2 cell.When ND effected for 12h,24h and 48h,the cell number in sub-G0/G1 peak(apoptotic peak) was increased gradually as time went on,the cell ratio of S decreased.4.The total protein level of ERK1/2 and AKT1 did not significantly change before and after potassium,chloride and calcium ion channel was blocked,but blocking potassium,chloride and calcium ion channel decreased phosphorylated protein level of ERK1/2 and AKT1,which showed obvious dose-effect relationship.Compared with control group,there were significant differences(p＜0.05).5.When TEA(5,10,15,20mM) blocked potassium ion channel,NPPB(50,100, 150μM) blocked chloride ion channel,ND(5,10,50,100μM) blocked calcium ion channel for 30min,1h and 2h.The relative expression of ADAR1 mRNA were decreased respectively time-effect and dose-effect dependently.6.Obvious correlation was found among Hep-2 cell proliferation suppressed by the blockers of ion channels(TEA,NPPB,ND),expression of phosphorylated protein level of ERK1/2,AKT1 and expression of ADAR1 mRNA.Third part The effects of blocking chloride ion channels on human laryngeal carcinoma xenograft tumors in nude miceMethods1.Hep-2 cells were used to establish laryngeal carcinoma nude mice xenograft models.The mice were divided into 5 groups after the neoplasm formed.The control group was treated with PBS,the treated groups with NPPB at different doses(10,50,100, 150μM).2.The nude mice's body mass and the tumors' size were determined termly to the painting growth curves of the xenograft models.After the treatment was ended,we stripped out the xenograft tumors and weighted.The inhibitory rate of xenograft tumors growth was figured out.3.TUNEL method was used to detecte the AI of the xenograft tumors.4.Western blot was used to detecte the phosphorylation levels of ERK1/2 and Akt1 protein in the xenograft tumors.5.The mRNA expression of ADAR1 in the xenograft tumors was analyzed by using RT-PCR.6.Statistical analysis:Statistical analyses were treated with the SPSS 13.0.t-test was used.The significant level was P＜0.05.Results1.When the treatment was ended,the tumor mass and the tumor size of the experimental groups(50,100,150μM NPPB) were significantly decreased(P＜0.05) comparing with the contrast group.2.The number of apoptotic cells in the experimental groups(50,100,150μM NPPB) were much more than that in the control group(P＜0.05).3.The expression of total protein of ERK1/2 and Akt1 in the xenograft tumors in the experimental groups(50,100,150μM NPPB) were not significantly(P＞0.05) changed. Compared with the contrast group,the phosphorylation levels of ERK1/2 and AKT1 in the experimental groups(50,100,150μM NPPB) were not significantly(P＜0.05) decreased. 4.The mRNA expression level of ADAR1 in the experimental groups(50,100, 150μM NPPB) was lower than that in the control group(P＜0.05).5.In the course of treatment,no adverse effects of NPPB were observed in the mice. The values of final body weight(FBW)/initial body weight(IBW) in five groups were more than 0.8 and it indicated that there was no toxic reaction in the mice.ConclusionsFor the first time,the effects of blockers of ion channels(TEA,NPPB and ND) on proliferation,apoptosis,cell cycle,and the phosphorylated ERK1/2,AKT1 protein levels,mRNA expression of ADAR1 in human laryngeal carcinoma Hep-2 cells were investigated by MTT,FCM,TUNEL,Western blot and RT-PCR in this study,and human laryngeal carcinoma xenograft tumors were also studied.Our conclusions were listed below:1.Hep-2 cell has voltage-dependent outwardly rectifying transmembrane potassium ion current.Chloride current of Hep-2 cell was outwardly rectifying,voltage-dependent and volume-sensitive.Calcium current of Hep-2 cell membrane was low-voltage activated.2.ADAR1 mRNA expressed in laryngeal carcinoma and corresponding para-carcinoma tissues.The expression of ADAR1 mRNA was irrespective with clinical stages,pathologic grade and metastasis of lymph.3.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could inhibit the proliferation,induce apoptosis of Hep-2 cells and this inhibitory effect showed time and dose dependence.The proliferation inhibition and apoptosis induction effects of TEA,NPPB and ND might be related to the cell cycle arrest at G0/G1 stage.4.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could down regulate the phosphorylation levels of ERK1/2 and AKT1 protein in Hep-2 cells in a time-dependent manner,which was relative with the proliferation of Hep-2 cells inhibited by TEA,NPPB and ND.5.Blockers of potassium,chloride and calcium ion channels(TEA,NPPB and ND) could inhibit the mRNA expression of ADAR1 in Hep-2 cells in a time-dependent manner. Down regulation of ADAR1mRNA expression was relative with inhibitory effect of TEA, NPPB and ND in the proliferation of Hep-2 cells.6.We established laryngeal carcinoma xenograft models successfully in nude mice,confirmed that NPPB(50,100 and 150μM) could inhibit the growth of xenograft tumors in dose-dependent manner.7.NPPB could down phosphorylation levels of ERK1/2 and AKT1 protein,induce cell apoptosis,and down regulating the mRNA expression of ADAR1 in laryngeal carcinoma xenograft tumors in nude mice.The mechanism of the effects might be related to down regulation of ERK1/2 and AKT1 protein,down regulation of ADAR1 mRNA expression.