| IntroductionPeritoneal metastasis is the commonest form of recurrence and the most important cause of death of advanced gastric cancer after radical dissection. It has been demonstrated that the interaction between cancer cells and extracellular matrix ( ECM) , which is mediated by various adhesion molecules, is one of the prerequisites for invasion and metastasis, among which CD44 and Integrinpl are two main and independent factors. CD44 is a family of glycoproteins on cell surface and plays various roles in physiological and pathological processes, including tumor metastasis, by recognizing its ligand, hyaluronic acid ( HA) , which is one of the main components of ECM. Integrinpl is a heterogeneous dimmer composed of a and p subunit, it can bind ECM protein and cytoskeleton at the same time to form a ligand -Integrinpl -cytoskeleton complex, mediating the adhesion and metastasis of tumor cells. The main ligand of Integrinpl is RGD, a tri -peptide composed of Arg - Gly - Asp and the functional structure of fi-bronectin. In this study, we investigated the anti - adhesive and anti - invasive effect of HA, Hyaluronidase ( Hase) , RGD and Integrinpl antisense oligodeoxy nucleotide ( asODN ) in order to find a feasible means of blocking the peritoneal metastasis of gastric cancer.MaterialsHuman gastric cancer line SGC7901. The main apparatus include incubator, Boyden chamber, FCM, PCR and electrophoresis apparatus etc. The main agents include DMEM, Matrigel, HA, Hase, RGD, mAb, oligodeoxynucleotide ( ODN ) , Lipofectin Reagent, TR-IZOL Reagent, Reverse Transcription System A3500, TaqDNA syn-thetase, Ultrasensitive S - P Kit etc.MethodsSGC7901 were cultured routinely with DMEM supplemented by 10% FCS. The expression of CD44 and Integrinpl on cell surface were determined by indirect fluorescence. After SGC7901 were treated by single factor or various combination of HA, Hase and RGD, their adhesion and invasion to ECM were measured by MTT and Boyden chamber method, respectively, the morphology of cells were also observed. Next, ODN were transfected into SGC7901, taking liposome as vector. The distribution of ODN was recorded by immunochemistry, changes in the expression of Integrinpl mRNA and protein were determined by RT - PCR and FCM, respectively. All the data were analyzed by SPSS10. 0 software.Results1. Human gastric cancer cell line SGC7901 did express CD44 and Integrin 1 protein on cell surface. 2. Hase or RGD alone couldblock the adhesion of SGC7901 to ECM( P <0. 001) , better than HA did ( P <0.05) , their inhibition on the invasion to ECM also had statistical significance ( p < 0. 05 ). Administration of HA and Hase in different sequence presented a better result in Hase + HA group than in HA + Hase group ( P < 0. 05 ). The inhibiting effect of the combination of the three factors was better than any one of the above groups ( P <0.001). 3. Observation on cell morphology revealed that the adhesive cells in treated group kept round while some of those in untreated group had spread out on Matrigel; in invasion assay, cells in untreated group presented a fibroid feature with pseudopod of various number and shape, while those in treated group kept round with relatively fewer pseudopod. 4. After Integrinpl asODN was transfected into SGC7901 it distributed evenly in cytoplasm and nucleus. PCR and FCM revealed a weakened band -on 489 bp and a shift - left curve, respectively, suggesting a decreased expression of Integrinpl mRNA and protein. 5. Adhesion and invasion assay also presented a decrease , with the inhibiting rate of 54% and 76%. The extent of decrease induced by Integrinpl asODN was larger than that by the random ODN (P<0.001).DiscussionThe invasion and metastasis of cancer is a multi - factor, multi -step and multi - stage process, and CD44 and Integrin 1 are two independent factors that modulating this process. In this study, we investigated the anti - adhesive and anti - invasive effect of HA, Hase, RGB and Integrinp 1 asODN in order to find a feasible means of blockin... |
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