Effects Of Catechin On Migration And Invasion Of Gastric Cancer Cells By Acid-sensing Ion Channels

Objective:Studies have shown that cancer incidence and mortality of gastric cancer is still high in all cancer diseases.In China,about two-thirds of patients with gastric cancer were diagnosed in the terminal stage of the tumor.Because it is not sensative to some conventional therapy methods,so surgery is still the main treatment method.But the tumor has the characteristic of invasion and metastasis,the main factors influencing the prognosis also include the invasion and metastasis of cancer,it is not only the root of the postoperative recurrence,but also makes many people lose the operation opportunity.Therefore the invasion and metastasis of cancer is still the key reason of its lethality,but the specific mechanism is unclear at present.ASICs is ion channel in the cells gated by proton.It was not only found in nerve cells,but also in Glioma cells and Salivary gland cancer cells,and ASICs has relationship with the invasion and metastasis of them.Catechin is a kind of flavanol compounds. Researches have shown that Catchin has function of anti-cancer and anti-mutation.it can reduce tumor angiogenesis and reduce the expression of E-cadherin to suppress the invasion and metastasis of cells.Interestingly,whether ASICs can effect on migration and invasion of gastric cancer cells, and whether Catechin can effect on it by ASICs or not.The aim of this study is to investigate the expression of ASICs in gastric cancer cells, the roles of ASICs in migration and invasion of gastric cancer by Transwell and Wound-healing experiment,and ASICs current by Whole-cell Patch Clamp,as well as the influence of Catechin on ASICs and migration and invasion of gastric cancer cells. Methods:1.Cultivate MGC803 and SGC7901 gastric cancer cells.2.Set up blank control group,Catechin50μmol/l 、 100μmol/l 、 200μmol/l 、400μmol/l goups. Detect the absorbance by MTT to observe the effect of Catechin on the survival rate of MGC803 and SGC7901 gastric cancer cells.3.Set up blank control group,Catechin 50μmol/l、100μmol/l、200μmol/l,Amiloride 50μmol/l、100μmol/l groups.Detect the expression of ASIC1 in every group by immunocytochemical technique.4.Set up blank control group,Catechin 50μmol/l、100μmol/l、200μmol/l,Amiloride 50μmol/l、 100μmol/l groups.Observe the migration ability in every group by Wound-healing Assay.5.Set up blank control group,Catechin 50μmol/l、100μmol/l、200μmol/l,Amiloride 50μmol/l、 100μmol/l groups.Observe the invasive ability of every group cell by Transwell Assay.6.Set up control group、Catechin 400μmol/l、Amiloride 100μmol/l groups,Observe ASICs current by Whole-cell Patch Clamp. Results:1.Detect the influence of Catechin on cell survival rate by MTT: Respectively set up four Catechin groups: 50μmol/l、100μmol/l、200μmol/l、400μmol/l groups,caculate the cell survival rate.In SGC7901 cells,respectly results were 90.90±1.275,77.78±3.298,64.78±3.309,26.06±6.440(%);In MGC803 cells,respectly results were 90.12±3.241、75.58±3.971、58.82±3.720、21.54±2.414(%).According to the results,we found that Catechin can reduce cell survival rate in MGC803 and SGC7901 cells.When the concentration reached 400μmol/l,the cell survival rate was dropped to as low as 50%.2.Detect expression of ASIC1 in every group by Immunocytochemistry technique: Expression of ASIC1 protein in SGC7901 cells was higher than that in MGC803 cells(P<0.05).Compared with the control group, 50μmol/l Amiloride can suppress the positive expression rate of ASIC1 in SGC7901 and MGC803 cells.(P<0.05), 100μmol/l Amiloride can significantly suppress the positive expression rate of ASIC1 in SGC7901 and MGC803 cells(P<0.01).In comparison with the control group respectively, 50μmol/l 、 100μmol/l Catechin can suppress the positive expression rate of ASIC1 in SGC7901 and MGC803 cells(P<0.05), 200μmol/l Catechin can significantly reduce the positive expression rate of ASIC1 in SGC7901 and MGC803 cells(P<0.01).The effect of Catechin on ASIC1 expression of gastric cancer cells was weaker than Amiloride(P<0.05).3.Detect the migration ability by Wound-healing Assay: The migration ability of MGC803 cells was weaker than that of SGC7901 cells(P<0.05).Compared with the control group, 50μmol/l Amiloride can reduce the migration ability of SGC7901 and MGC803 cells( P<0.05),100μmol/l Amiloride significantly reduced the migration ability of SGC7901 and MGC803 cells(P<0.01),so inhibiting the expression of ASIC can inhibit the migration ability in MGC803 and SGC7901 cells.In comparison with the control group respectively, 50μmol/l、100μmol/l Catechin can suppress the migration ability of SGC7901 and MGC803 cells(P<0.05), 200μmol/l Catechin significantly suppressed the migration ability of SGC7901 and MGC803 cells(P<0.01). The effect of Catechin on migration of gastric cancer cells was weaker than Amiloride(P<0.05).4.Detect the invasive ability by Transwell Assay: The invasion ability of MGC803 cells was weaker than that of SGC7901 cells(P<0.05).Compared with the control group,50μmol/l Amiloride can reduce the invasive ability of SGC7901 and MGC803 cells(P<0.05),100μmol/l Amiloride significantly reduced the invasive ability of SGC7901 and MGC803 cells(P<0.01),so inhibiting the expression of ASICs can inhibit the invasive abilty of MGC803 and SGC7901.In comparison with the control group respectively,50μmol/l 、 100μmol/l Catechin can reduce the invasive ability of SGC7901 and MGC803 cells(P<0.05), 200μmol/l Catechin significantly reduced the invasive ability of SGC7901 and MGC803 cells(P<0.01). The effect of Catechin on invasion of gastric cancer cells was weaker than Amiloride(P<0.05).5.Detect the ASICs current by Whole-cell Patch Clamp: In MGC803 and SGC7901 cells we can detect ASICs current,ASICs current of MGC803 cells were lower than ASICs current of SGC7901 cells( P<0.05).After 100μmol/l Amiloride(ASICs blocker)was added,the current was inhibited(P<0.05).Compared with the control group, 400μmol/l Catechin can also inhibit ASICs current in MGC803 and SGC7901 cells(P<0.05),but the effect of Catechin on ASICs current was weaker than Amiloride(P<0.05). Conclusions:1.ASICs may take part in the migration and invasion of gastric cancer.2.Catechin can inhibit the migration and invasion ability of MGC803 and SGC7901 cells,this effect may have relationship with effect of Catechin on ASICs.