Changes Of Multiple Potassium Channels In Gastric Cancer Cells And Their Role In Proliferation

Gastric cancer（GC）is a common tumor of the digestive tract system and one of the important cancers that threaten human health.The mortality rate ranks second among all cancers in China^[1].Surgery is the main treatment for patients with gastric cancer.In recent years,the level of surgery has been greatly improved,and combined with adjuvant therapy such as chemotherapy,radiation therapy and molecular targeted therapy,the cure rate has been greatly improved.However,the incidence and overall survival rate of gastric cancer patients have not been significantly improved.The main reason for this situation is that the molecular mechanism of the occurrence and development of gastric cancer is still unclear,so it is impossible to explore effective treatment methods^[2].About 13%of the effective drugs for clinical treatment of tumors are directed to ion channels.Potassium ion channels are the most classified and most complex type of channels among various ion channels.Abnormal potassium channels can cause membrane potential imbalance,epilepsy,arrhythmia,and so on.More and more studies have found that abnormal expression of potassium channels are associated with tumors.At present,potassium channel is mainly divided into four types:calcium-activated delayed rectifier potassium channels（KCa）,voltage-dependent potassium channels（Kv）,inward rectifier potassium channels（Kir）and double-pore potassium channels（K2P）.Objective:This experiment compared the characteristics of potassium currents mediated by different types of potassium channels in normal gastric epithelial cells（GES-1）and gastric cancer cells（HGC-27 and AGS）to understand the types of potassium channels that play a major role in gastric cancer cells.Further detection and analysis of the differences in expression and function of the main channels between gastric cancer cells（HGC-27 and AGS）and normal gastric epithelial cells（GES-1）.It is hoped that by studying the relationship between gastric cancer development and potassium channels,it will provide a new scientific basis for explaining the pathogenesis of gastric cancer and targeted therapy.Methods:（1）Using the whole-cell patch-clamp technique,four kinds of channel-specific inhibitors were added to record four kinds of potassium currents mediated by different potassium channels between normal gastric epithelial cells（GES-1）and gastric cancer cells（HGC-27 and AGS）,including KCa channel current I_KCa,K2P channel current I_K2P,Kv channel current I_Kv,Kir channel current I_Kir.（2）Using Q-PCR technology to observe the potassium channel m RNA expression in normal gastric epithelial cells（GES-1）and gastric cancer cells（HGC-27 and AGS）.（3）Using fluorescence imaging technology to detect the difference of Intracellular free calcium concentration([Ca²⁺]_i)in normal gastric epithelial cells（GES-1）and gastric cancer cells（HGC-27 and AGS）.（4）The cell viability assay（MTS）was used to detect the difference in the proliferation of potassium channels to normal gastric epithelial cells（GES-1）and gastric cancer cells（HGC-27 and AGS）.Results:（1）KCa channels,Kv channels,Kir channels and K2P channels specific inhibitors TEA（2.5 m M）,4-AP（10 m M）,Ba Cl₂（5μM）and Propafenone（100μM）can respectively inhibit the potassium current caused by the step pulse,suggesting that there were four different potassiums channel-mediated potassium currents in the three cells;（2）In GES-1,potassium channels were abundant,and the potassium currents showed a trend of I_KCa=I_K2P>I_Kv=I_Kir;in HGC-27,the potassium currents showed a trend of:I_KCa>I_Kv=I_K2P=I_Kir;in AGS,the potassium currents showed a trend of I_KCa>I_K2P>I_Kir=I_Kv.It was suggested that the KCa channel was the main potassium channel among the four types of potassium channel in the three types of cells,playing a leading role;（3）The potassium currents of gastric cancer cells HGC-27 and AGS were significantly lower than those of GES-1（P<0.01）;（4）KCa played a leading role in multiple cells.When the[Ca²⁺]_i level was low,I_KCa in GES-1 was significantly higher than HGC-27 and AGS.A23187（1μM）was further added to these three types of extracellular fluids to increase the[Ca²⁺]_i to high calcium levels.It could be observed that the I_KCa caused by these three types of cells increased significantly（P<0.01）,the I_KCa in GES-1 was still significantly higher than that in HGC-27 and AGS;（5）The results of Q-PCR showed that KCNMB1/3/4 and KCNN2/4 in gastric cancer cells were lower（P<0.05）,KCNN1was increased（P<0.05）,and KCNMB2 was missing in all three types of cells compared with normal gastric epithelial cells in GES-1.It was suggested that the KCa channels of gastric cancer cells was generally under-expressed,which was consistent with patch clamp results;（6）Keeping the cells insided the high potassium state（40m M）,the inhibition rate of the GES-1 group was 28.06%±0.53%（n=3）,higher than the HGC-27 group of 15.33%±3.49%（n=3,P<0.01）and AGS group of 14.6597%±4.60%（n=3,P<0.01）,suggesting that high potassium administration can inhibit the proliferation of three kinds of cells,and the degree of inhibition was weak;（7）After TEA inhibited KCa channels,the inhibition rate of the GES-1 group was 36.54%±0.57%（n=3）,HGC-27 group:12.38%±1.39%（n=3,P<0.01）,AGS group:21.56%±1.97%（n=3,P<0.01）,the degree of inhibition was GES-1>AGC>HGC-27,suggesting that inhibition of KCa channels can significantly inhibit cell proliferation;（8）Nifedipine（NIF）inhibited voltage-dependent calcium channels（L-VDCC）resulting in less calcium influx,[Ca²⁺]_i decreases,and inhibited the proliferation of gastric cancer cells.The inhibition rate of GES-1 group was 58.31%±0.54%（n=3）,HGC-27 group:60.83%±2.18%（n=3,P>0.05）,AGS group:64.88%±3.51%（n=3,P>0.05）,suggesting that NIF can significantly inhibit the proliferation of three types of cells.There was no difference in its degree of inhibition.Conclusion:There are four different kinds potassium channels in normal gastric epithelial cells（GES-1）and gastric cancer cells（HGC-27 and AGS）,and the functions of the four potassium channels in gastric cancer cells are all weakened;It plays a leading role,and the m RNA expression of KCa channels in gastric cancer cells is reduced,Inhibiting KCa channel can inhibit cell proliferation;High potassium inhibits the proliferation of three types of cells.