| 【Background】Gastric Cancer is one of the most common malignant cancers in China. Distantmetastasis accounts for>90%of mortality from gastric cancer and unfortunately,currently no effective means capable of interdicting and perhaps even reversing theprocess of metastasis is available. Genistein is a small natural compound extracted fromsoy products, with an antioxidant and a weak estrogen mimic activity. Epidemicalinvestigation as well as basic and clinical study results suggested Genistein is able toreduce the motility of cancer cells, especially in the estrogen dependent prostate cancerand estrogen dependent breast cancer, resulting in decreased metastasis related motality.So far, whether Genistein affects the invasive and metastatic ability of gastric cancerremains unknown.Genistein’s estrogen like effect, due to the similarity between Genistein and17-β-estradiol, together with the nonspecific bone marrow toxicity limited the clinicalapplication of this compound. To overcome Genistein’s side effects, several compoundswith–OH in Genistein substituted with other groups were developed by our group. Finally, a Fluorine substituted molecule was found to with much less toxicity while remaining theanti-metastasis property.Metastasis cascade is a complex process but the currently used metastatic modelssuch as the Transwell assay,the wound healing assay and the tail vein metastasis animalmodel usually just represent one or several stages of the process, therefore morecomprehensive model is needed. Orthotopic implantation model is a better choice in that itmimics the whole process of metastasis, from the local proliferation to intravasate into thelumina of blood vessels to survive the rigors during circulation to extravasate into a distantorgan and form a new colony.【Objectives】This study was designed to systemically investigate the anti-metastatic effects ofGenistein and KBU2046in gastric cancer; to explore the mechanisms underlying theanti-metastatic effects of Genistein and KBU2046; and to establish an orthotopicimplantation gastric cancer model, seting the foundation for further investigation.【Methods】A. The effects of Genistein and KBU2046on the proliferation of gastric cancer cells wasmeasured using the MTT method; the effects of Genistein and KBU2046on cell cycleprogression and apoptosis was detected using flow cytometry.B. The effects of Genistein and KBU2046on the migration and invasion ability wasevaluated with the Transwell assay; The effects of Genistein and KBU2046on the cellmigration was dynamically recorded with cellomics platform.C. Changes of metastatic related gene expression in gastric cancer after exposure toGenistein and KBU2046were screened using PCR Array. The screening results were thenfurther validated using real time quantitative PCR and Western blotting.D. Luciferase labeled gastric cancer cells were injected through the tail vein, Genisteinand KBU2046were intragastrically administrated daily. The metastatic foci wereevaluated using the living animal imaging system.E. Subcutaneously planted gastric cancer tissue were dissected and cut into small pieces. Small tissue blocks were orthotopically planted The seromuscular layer of the greatercurvature of stomach. Since4thweek,3mice were sacrificed every week. Stomach, liverand lung of the model animal were removed and HE staining were performed.【Results】（1） KBU2046did not affect the proliferation of gastric cancer cells and immortalizedgastric epithelial cells even at the concentration of50uM. The proliferation of gastriccancer cells exposed to50uM Genistein were inhibited after24h and the proliferationof gastric cancer cells and immortalized gastric epithelial cells exposed to12.5uMGenistein were both inhibited after72h. Exposure to10uM Genistein and KBU2046has no effect on cell cycle progression and apoptosis of gastric cancer cells.（2） Transwell assay and cellomics data suggested that both Genistein and KBU2046reduced the migration and invasion ability of gastric cancer cells, but did not affectthe motility ability of immortalized gastric epithelial cells.（3） We used the PCR Array technology to screen the changes in the expression of120metastasis related genes in gastric cancer cells before and after Genistein andKBU2046exposure.12and9genes were changed with more than2fold changesafter Genistein and KBU2046treatment respectively. We focused on MMP2, MMP7,IGF1and CTSK and real time quantitative PCR results confirmed that Genistein andKBU2046exposure decreased the mRNA level of the four genes, with MMP7beingmost significantly affected. Western blotting result showed that MMP7expressionwas also decreased at the protein level.（4） Images acquired in the living animal imaging system showed that metastaticsignals in the nude mice treated with25mg/kg and75mg/kg body weight were muchless compared with the control group. And a more remarkable effect were observed inthe75mg/kg group. The treatment did affect the body weight of the tumor bearingnude mice.（5） The imaging signal increased with the time progression and HE staining slicesconfirmed the tumor located between the mucosa and serosa layer. Metastatic signals were detected in the lung tissue but not the liver tissue..【Conclusion】In the present study we systemically investigated the effects of Genistein andKBU2046treatment on the migration and invasion ability of gastric cancer cells. Theresults showed that both Genistein and KBU2046inhibited the migration and invasionpotential of gastric cancer cells with affecting cycle progression and apoptosis. Primarymechanism study showed that Genistein and KBU2046exposure downregulated severalmetastasis related gene expression, with MMP7displayed the most significant fold change,suggesting that Genistein and KBU2046might exerted their effect through targetingMMP7. Finally, we successfully established an orthotopic implantation gastric cancermodel, which may be applied for study of gastric cancer metastasis. |
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