Study On Anti-metastsis Mechanism Of Qigefang In Gastric Cancer

Gastric cancer is a common malignant tumor. As the highest incidence tumor in alimentary system, it's incidence accounts 62% in alimentary system tumor. The incidence of gastric cancer in our country is high and far higher than western country. About 170 thousand people died of gastric cancer,it takes 50% death number in alimentary system tumor in our country every year. With increasing medical level and applying comprehend treatment,5 year survival rate of gastric cancer has improved,but overall therapeutic effect is still unsatisfactory. The reason is there is non-typical symptom of early gastric cancer, most patients with progression caner when they were diagnosed. The metastasis always causes treatment failure after surgery. It is reported about 40%-60% of patients with gastric cancer recurrence and metastasis. To seek reliable methods to treat gastric cancer metastasis is an urgent problem we are facing.The primary route of metastasis is blood metastasis, lymphatic metastasis, peritoneum implantation metastasis.Perniciousness of blood route metastasis is the largest dangerous.By the blood circulation gastric cancer-cell can form metastsis in other organ and destroy organ's function to lead to die. Liver and lung is the most vulnerable organs to the transfer of blood. It is reported liver metastsis incidence is 40-50% and lung metastsis is 40% in gastric cancer.Chemotherapy is the main therapy after liver and lung metastsis.Although combined with chinese medicine,radiotherapy,operation and immunotherapy median-survival time is only 6-9 months. Therefore, metastasis preventing is key point on cure gastric cancer and important sense to increase survival time. The hot-spots in tumor research are mechanism of tumor metastasis and anti-metastasis medicine. The basic process of tumor metastasis has probably clear. Anti-metastasis medicine concentrate on anti-new-vessels, anti-platelet aggregation, anti-platelet activation, anti-metastasis gene, raising immune function, which most are in experimental stage. Moreover, the metastasis of gastric cancer is multi-factor, multi-step, and multi-link process, while these medicines'single channel and target lead to the role in inhibiting metastasis utterly inadequate. Complex prescription of chinless medicine is reasonable combination based on"planning treatment according to Diagnosis". It has much complex composition and has many curing function. Thus, we researched anti-metastasis function of Qigefang on the basic process of metastasis and correlated factors.Qigefang changed from Qigesan in <> which Qigesan was used to treat Dysphasia. While treating patients with gastric cancer and esophageal cancer, we find Qigefang can prolong life span and prevent or delay metastasis of gastric cancer in advanced stage. Qigefang can prevent metastasis to prolong disease free survival date (DFS) used for patients after they were operated. Therefore we think Qigefang has anti-metastasis function on gastric cancer. The research investigates Qigefang's effective target and effective link on restraining metastasis from process blood route metastasis of gastric cancer.We observed anti-metastasis function of Qigefang in vitro test and in vivo test. After cultured high-invasion cell line of gastric cancer and esophageal cancer, we observed the influence of Qigefangon on metastasis ability of tumor cell. In vivo test, mice were vaccinated by high-lung metastasis gastric cancer cell line, we observed inhibition ratio of lung metastasis and influence on related metastasis factor.The research includes four parts:Part one: Effects of Qigefang on adhesion of human gastric cancer cell MGC and of human esophageal cancer cell EC109Objective: To observe Qigefang's effect on homogeneous adhesion between gastric cancer cell line MGC and gastric cancer cell line MGC, between human esophageal cancer cell line EC109 and human esophageal cancer cell line EC109. To detect difference of adhesive ratio, aggregate formation ratio and E-Cadherion expression among Qigefang group, 5FU group and control group by several methods.Methods: Resused human gastric cancer cell line MGC and human esophageal cancer cell line EC109 were devived randomly into five groups when cell was at logarithmic growth phase. Add Qigefang in Chinese medicine group to make final concentration be 15mg/ml,30mg/ml,60mg/ml and called low-dose group,meta-dose group, macro-dose group; Add 5FU chemothapy group to make final concentration be 50ug/ml;add PBS in control group.To detect adhersion status at 20min,40min,60min of each group by machine method and to compute adhersion ratio. To detect intensity of radiation at 30min,60min by active nucleus and to compute adhersion ratio. To detect formation of aggregate at 2h,4h,6h,12h and to compute formation ratio of aggregate and measure the largest area of aggregate in each group.To measure expression of E-cadherin by flow cytometry.Results:To detect adhersion ratio of gastric cancer cell MGC and esophageal cancer cell EC109 at 20min,40min,60min by machine mothod ,each dose group of Qigefang was higher than 5FU group and control group.The difference between Qigefang group and 5FU group or control group was significant(P<0.01). The difference between 5FU group and control group was not significant(P>0.05). The difference among each dose group of Qigefang was not significant(P>0.05). By active nucleus to detect adhersion ratio of gastric cancer cell MGC and esophageal cancer cell EC109 at30min,60min ,each dose group of Qigefang was higher than 5FU group and control group.The difference between Qigefang group and 5FU group or control group was significant(P<0.01). The difference between 5FU group and control group was not significant(P>0.05). The difference among each dose group of Qigefang was not significant(P>0.05). The largest area of aggregate of MGC and EC109 in each dose Qigefang group was larger than control group and 5FU group (P<0.05), 5FU group was smaller than control group.Expression of E-cadherin in each Qigefang was higer than control group and 5FU group by flow cytometry. The difference between 5FU group and control group was not significant(P>0.05), The difference among each dose group of Qigefang was not significant(P>0.05).Conclusion: Qigefang could raise adhersion ratio,formimg ratio of aggregate of human gastric cancer cell line MGC and human esophageal cancer cell line EC109.Qigefang could raise homogeneous adhersion capability.5FU had not obvious influence about homogeneous adhersion ratio.when 5FU effected tumor cell for a long time,it would retrain aggregate form. Qigefang could raise E-cadherin expression of human gastric cancer cell line MGC and human esophageal cancer cell line EC109. Part two: Effects of Qigefang on cell proliferation,movement,secreting MMP-2 and MMP-9 of of human gastric cancer cell MGC and of human esophageal cancer cell EC109Objective: To observe that Qigefang had inhibitory action on cell proliferation,movement,secreting MMP-2 and MMP-9 of MGC and EC109 cell line.Compare with 5FU group,found the difference in cytostasis ratio,moving speed,enzyme activity of MMP-2 and MMP-9.Methods:To detect tumor cell proliferation by MTT mothod,100ul Cell suspension was added in each hole of 96 orifice(each hole 2×105 cell)and tumor cell was divided into Qigefang group,5FU group and control group.Qigefang were added into Qigefang group to make final concentration be 15mg/ml,30mg/ml,60mg/ml and called low-dose group,meta-dose group, macro-dose group. 5FU were added into 5FU group to make final concentration be 50ug/ml. PBS was added into control group.Incubated 24,48,72h in incubaton and added MTT 4h before incubation accomplished.Detect OD value in each time spot and count proliferation inhibition ratio. To detect moving ablity of tumor cell by agar drop mothod.Made agar suspension of MGC,EC109 cell and added 100ul in each hole of 24 orifice to detect basic radius after cooling congelation at 4℃.Divided into Qigefang group,5FU group and control group. Medical concentration of each group were similar to MTT method.To measure movement radius of each group cell and count moving speed of each group cell.To detect MMP-2,MMP-9 activity by gelatinase zymography .Groups were same as above.To obtain supernatant after cultured 24h and made polyacrylamide gel electrophoresis.To analyze MMP-2,MMP-9 activty based on strap gray scale.Results: Qigefang had obviously depressant effect on vitro-proliferation of MGC,EC109 cell.It's depressant fuction became more obviously with raising medicine density. Macro-dose of chinese medicine had the strongest depressant effect on tumor cell.Inhibition ratio had reach 60.0% .The highest depressant ratio was 77.1% after cultured 72h with Chinese medicine. The difference among each-dose of Chinese medicine group ,5FU group and control group was all significant ( P<0.05 ) . The difference between Macro-dose of Chinese medicine group and 5FU group was not significant(P>0.05).To detect moving action of each group cell was all poor when cultured at 12h. Moving ablity of control group became strong obviously and moving speed was faster than Qigefang after cultured 24h.Control group showed strong locomotor activity. locomotor activity of 5FU group was also stronger than Qigefang group, The difference was significant(P<0.05). The difference among each dose group of Qigefang was not significant(P>0.05).Control group cell secretion of MMP-2,MMP-9 was more than Qigefang group and 5FU group(P<0.05).MMP-2 was stronger obviously(P<0.01).Activity of MMP-2 and MMP-9 in Qigefang group was waker than 5FU group. The difference was significant(P<0.05)and the difference between each-dose of Chinese medicine was not significant(P>0.05).Conclusion: Qigefang could suppress reproductive activity of MGC and EC109 cell, Macro-dose compare with 5FU the difference was not significant. Qigefang could suppress moving ablity of MGC and EC109 cell and slow down moving speed. Cell locomotor activity of 5FU group was stronger Chinese medicine group.Qigefang could suppress activity of MMP-2 and MMP-9 which tumor secreted.Enzymatic activity of 5FU was stronger than Qigefang group.Part three: Inhibiting effects of Qigefang on lung metastsis of mice with gastric bearing cancerObjective: To observe Qigefang effect on tumor growing of mice with gastric bearing cancer. To observe Qigefang depressant function on pulmonary metastsis of mice with gastric bearing cancer by counting pulmonary metastsis nodus and to compare 5FU depressant function on pulmonary metastsis of gastric bearing cancer of mice.Mothods: To obtain cell suspension of gastric cancer to vaccinate at right armpit hypo of 30mice 615, a mouse 0.1ml(1×10 6cell).Divided into three groups randomly------Qigefang group, 5FU group,control group.Each group was 10 mice. After vaccinated 3 days,the mice in Chinese medicine group were drenched Qigefang fluid 0.2ml(0.3g)each day. the mouse in chemotherapy group were injected 5FU 0.2ml(200ug) in abdominal cavity. the mouse in control group were drenched isotonic Na chloride 0.2ml.After 16 days killed the mouse,stripped the tumor architecture,weighed tumor,measuse tumor diameter,compute gross tumor volume.Dislodged lung architecture and fixed by 10% neutro formalin.To observse metastasis on lung architecture,compared quantity of metastasis nodus,computer depressant ratio of pulmonary metastsis.Paraffin imbedding,cut sheet(thickness 4um),HE drum dyeing commonly,compare metastasis degree in lung with each group.Results: Tumor weight ,tumor diameter and tumor volume of Chinese medicine group and chemothapy group were obviously lower than control group(P<0.01).The difference between Chinese group and chemotherapy group was not significant(P>0.05).Mouse of control group 100% happened lung metastsis by directly counted white metastsis nodus on lung surface.Number of metastsis nodus on lung were less than control group's and metastsis degree were less than control group obviously(P<0.01).There were 4 mice no metastsis nodus(P<0.01 ).Compared with chemotherapy group, metastsis nodus on lung and inhibition ratio of lung of Chinese medicine group was better than chemotherapy and there was difference of metastsis degree between Chinese medicine group and control group . There was a mouse no metastsis nodus in chemotherapy group.The difference of number of metastsis nodus,lung metastsis degree and inhibition ratio of lung metastsis between chemotherapy group and control group was significant(P<0.05).Conclusion: Qigefang could inhibit tumor growing of mice model of gastric cancer,decrease pulmonary metastsis,lessen lung metastsis degree.5FU also could decrease pulmonary metastsis and lessen lung metastsis degree,but inhibitory action on metastsis inferior to Qigefang.Part four: Effects of Qigefang on gastric cancer metastsis correlation factorObjective: To observe Qigefang effect on adhesion molecule E-cad ,Proteo lytic enzyme MMP-2,MMP-9,blood vessel endothelial growth factor VEGF,microvessel density MVD of transplantation tumor of mouse gastric cancer.To detect blood plasm level of GMP-140, GMP-140 of mice . and find difference among groups.Mothods: Grouping,making model,pouring medicine were similar to experiment in part three .Removal eye globe to obtain blood,gained blood plasm for pre-emergency,killed the mouse,stripped tumor architecture. 10% neutro formalin fixed. Paraffin imbedding,cut sheet(thickness 4um).To detect expression of E-Cad,MMP-2,MMP-9,VEGF,MVD on tumor of each group by SP immunohistochemistry.To detect GMP-140 level of mouse blood plasm by radio-immunity.To detect GMP-140 level of mouse blood plasm by euzymelinked immunosorbent assay.Results: There were different degree expression of E-cad on tumor architecture of each group.Expression of E-cad of Chinese medicine group was stronger than control group and 5FU group. The difference was significant(P<0.01). The difference between control group and 5FU group was also significant(P<0.05).MVD of control group was obviously higher than Chinese group and 5FU group(P<0.05). The difference between Chinese medicine group and 5FU group was significant(P<0.05). Expression of VEGF of Chinese medicine group and 5FU group was lower than control group. The difference was significant(P<0.05). The difference between Chinese medicine group and 5FU group was not significant(P>0.05). There was different degree expression of MMP-2,MMP-9 on tumor architecture of each group. Expression of MMP-2,MMP-9 of Chinese medicine group was lower than contrl group, The difference was significant(P<0.05).Expression of MMP-2,MMP-9 of 5FU group was also lower than control group, The difference was significant(P<0.05). The difference between Chinese medicine group and 5FU group was not significant(P>0.05). level of GMP-140 and TXB2 of mouse blood plasm Chinese medicine group and 5FU group was lower than control group obviously, especially level of blood plasm GMP-140, The difference among Chinese medicine group ,5FU group and control group was highly significant(P<0.01),the difference between Chinese medicine group and 5FU group was not significant. level of blood plasm TXB2, the difference between Chinese medicine group and 5FU group was significant(P<0.05).Conclusion:Qigefang can raise expression of adhesion molecule E-cad, depress expression of proteolytic enzyme MMP-2 and MMP-9, depress expression of blood vessel endothelial growth factor VEGF, depress microvessel density MVD, depress GMP-140,TXB2 level of mouse blood plasm.Therefor,Qigefang has inhibiting effection on metastsis.