Effect Of Interleukin8 On Gastric Cancer Cell Invasion And Intervention Study Of Xiaotan Sanjie Decoction

BackgroundGastric cancer is one of the most frequent tumors in china. Pathologic character of gastric cancer is easy to invade and metastasize which is one of the reasons for difficult treatment and bad prognosis. The clinical researchs find out that IL-8 expresses high in tumor issue and blood and the expression level is correlated with infiltrating depth, lymph node metastasis and prognosis in recent years. Professor Wei-Pinkang has established his theory of"dispersing phlegm and dissipating bind"for treating patients with gastric carcinoma and other tumors clinically. The therapeutic effect is satisfactory. Therefore, the purpose of this study is to discover the mechanisms of IL-8 on promoting gastric cancer cells to invade by detecting effects of IL-8 on adhesion, invasion and degradating extracellular matrix. And Xiaotan Sanjie Decoction is used in vivo and vitro to observe its effects on IL-8 expression and regulation in invasion about gastric cancer and discover possible function of Xiaotan Sanjie Decoction on resisting invasion.ObjectiveThe study was designed to investigate the mechanism of IL-8 on invasion in gastric cancer by culturing human gastric cancer cell SGC7901 in vitro with recombinant human IL-8 and detecting adhesive and invasive abilities and expression of matrix metalloproteinase and adhesion molecule. Medicine serum of Xiaotan Sanjie Decoction was used to interfere in gastric cancer cell cultured with rIL-8 and Xiaotan Sanjie Decoction was used to mouse bearing cancer.The changes of adhesive and invasive abilities and the expression of IL-8 and its receptor were detected to discover possible function of Xiaotan Sanjie Decoction on resisting invasion.Methods1. Effect of IL-8 on gastric cancer cell invasion:(1) ELISA was used to detect IL-8 protein in gastric cancer cell SGC7901 in 24h,48h and 72h respectively.(2) Different concentrations of rIL-8(0ng/ml,1ng/ml,10ng/ml,50ng/ml,100ng/ml) were used to culture gastric cancer in vitro and cell proliferation and adhesion to blood vessel endothelium cell and ECM were detected by cck-8; Scratch - repair experiment was used to detect cancer cell migrative ability;The invasive ability was observed by Transwell;The protein and gene expressions of MMP-2,MMP-9,E-cad and ICAM-1 were observed by Westenblot and RT-PCR. 2. Intervention Study of Xiaotan Sanjie Decoction on IL-8 in gastric cancer cell invasion:(1) Xiaotan Sanjie Decoction -containing serum was made. CCK-8 was used to observe the effect of Xiaotan Sanjie Decoction on IL-8 expression.(2) Xiaotan Sanjie Decoction -containing serum was used to interfere in gastric cancer cell cultured with rIL-8(100ng/ml). Adhesion to blood vessel endothelium cell and ECM were detected by cck-8; Scratch - repair experiment was used to detect cancer cell migrative ability;The invasive ability was observed by Transwell; The protein and gene expressions of MMP-9, E-cad and ICAM-1 were observed by Westenblot and RT-PCR.3. Effect of Xiaotan Sanjie Decoction on expression of IL-8 and its receptors in human gastric transplanted tumor model:Nude mouse were used to make human gastric transplanted tumor model subcutaneouly and randomly divided into control group and Xiaotan Sanjie Decoction group; The mice bearing cancer were given intragastric administration 48 hours after the surgery ( 0.4ml, once a day ,6 times a week×4w);The changes of all over the body and body weight were obversed; The experiment was terminated 4 weeks later and the mouse were sacrificed.Tumor issue was got and weighed;gastric cancer ultramicrostructure was obversed by transmission electron microscope and the protein expression of IL-8, its receptors (CXCR1and CXCR2)and MVD(CD34marking)level was detected byELISA and SABCimmunohistochemistry.Results1. The IL-8 protein expressed in human gastric cancer cell and IL-8 concentration was increased according to the time increasing.IL-8 protein was (277.476±26.650)pg/ml when cancer cells were cultured for 72h and compared with 24h and 48h groups, there was significant difference.2. The CCK-8 results shown that rIL-8 had no effect on SGC7901cell proliferation(P=0.995); rIL-8 could promote human gastric cancer cell to adhere to endothelial cell and there was statistical significance between different groups(P <0.01).Also rIL-8 could promote human gastric cancer cell to adhere to EMC(collagen, laminin and fibronectin)and there was statistical significance between different group(sP <0.01,P <0.01,P <0.01). The adhesive abilities were significantly increased in a rIl-8 concentration dependent.3. The results of scratch - repair experiment shown that rIL-8 could significantly promote cells migrate into the cell-free region in 24h and the migrative ability was increased gradually with the dose increasing of rIl-8; The Transwell results shown that rIL-8 significantly accelerated gastric cancer cells invasion and cell number through Matrigel ECM membrane was added.There was statistical significance between different groups (P <0.01).4. Westenblotand RT-PCR results shown: rIl-8 did not significantly influent the MMP-2 expression level under the tested conditions; but protein and mRNA expression levels of MMP-9 and ICAM-1 were increased gradually with the dose increasing of rIl-8 and E-cad was decreased gradually. There was a dose-dependent relationship.5. Xiaotan Sanjie Decoction -containing serum could decrease IL-8 protein and there was statistical significance between control and blank-containing serum(P <0.01, P <0.01). IL-8 protein in blank-containing serum group was more than that in control group(P <0.05); Xiaotan Sanjie Decoction -containing serum could inhibit cell proliferation compared with control and blank-containing serum groups(P <0.01,P <0.01).6. rIL-8(100 ng/ml)was used to culture human gastric cancer cell SGC7901 and interfered by Xiaotan Sanjie Decoction -containing serum:(1) Xiaotan Sanjie Decoction -containing serum could inhibit the effect of rIL-8 on cancer cell to adhere to endothelial cell and ECM components (collagen, laminin and fibronectin) and had significant difference with controland blankgroups.(2) Xiaotan Sanjie Decoction -containing serum could inhibit the effect of rIL-8 on the migrative ability in 24h.The Transwell results shown that Xiaotan Sanjie Decoction-containing serum significantly decelerated gastric cancer cells invasion and cell number through Matrigel ECMmembrane affected by rIL-8, compared with controland blank-containing serum groups (P <0.01,P <0.01).(3) Xiaotan Sanjie Decoction -containing serum could inhibit the effect of rIL-8 on expression levels of MMP-9, ICAM-1 and E-cad.7. Effects of Xiaotan Sanjie Decoction on growth of human gastric transplanted tumor: There were transplanted tumors about all 20 nude mouse in right armpit. The weight change subtracting tumor weight in control group had no statistically significance with that in Xiaotan Sanjie Decoction group(P>0.05); tumor weight in Xiaotan Sanjie Decoction group is smaller that in control group(P <0.05).8. Effects of Xiaotan Sanjie Decoction on gastric cancer ultramicrostructure: To observe gastric cancer issiue by transmission electron microscope: chromatin in control group was spissated and annuliform, there was little vacuole in kytoplasm, cell envelope was complete, there was little parapodium, ECM structure was damaged seriously, cystoskeleton was loose and basement membrane was dissolved. In Xiaotan Sanjie Decoction group, cell nucleus was pyknosis and in the state of clumping distribution and apoptosis, there was no obvious parapodium and ECM structure was damaged moderately.9. Effects of Xiaotan Sanjie Decoction on expression of IL-8,its receptors and MVD: Xiaotan Sanjie Decoction could down-regulate the expressions of IL-8 and its receptors(CXCR1 and CXCR2)protein compared with control (P <0.01);MVD number in Xiaotan Sanjie Decoction group was lower than that in control group (P <0.01); Dependablity ofIL-8andMVD: there was dependability between IL-8 protein and MVD number in control(r=0.788,P=0.007).Conclusion1. IL-8 can promote adhesive and invasive abilities of human gastric cancer cell SGC7901and regulate the expression of MMP-9, E-cad and ICAM-1. This might be one of the mechanisms promoting gastric cancer invasion.2. Xiaotan Sanjie Decoction-containing serum can inhibit IL-8 protein expression and interfereobviously in effect of IL-8 on adhesive and invasive abilities of human gastric cancer cell and the expression of MMP-9,E-cad and ICAM-1; Xiaotan Sanjie Decoction can inhibit the growth of human gastric transplanted tumor model subcutaneouly and downregulate the expression of IL-8 and its receptors (CXCR1andCXCR2) and MVD number.3. Xiaotan Sanjie Decoction can regulate expression and effect of IL-8 and its receptors which has a strongly effect on gastric cancer cell invasion. This might be one of the mechanisms of Xiaotan Sanjie Decoction resisting invasion.