Mechanism Of SCIN-promoting Invasion In Gastric Cancer Cells And Establishment And Characterization Of A New Gastric GIST Cell Line

Part? Study on the mechanism of SCIN-promoting invasion in gastric cancer cellsBackground and objective:Gastric cancer(GC)is the fourth-most prevalent cancer worldwide and the second-most common cause of cancer-related death.Although the mortality rate has been decreasing over recent years due to significant improvements in early diagnosis,surgical techniques and chemotherapy,the long-term survival rate for patients with advanced-stage GC still remains low.Many GC patients suffer from recurrence or metastasis after radical operation and chemotherapy,leading to a more complicated situation for further treatment and reoperation.Invasion and metastasis are important properties of GC that contribute to poor prognosis of patients and the underlying mechanisms still remain unclear.Therefore,better understanding of the molecular mechanisms underlying invasion and metastasis of GC is crucial for the effective treatment.Our previous work has adequately demonstrated that Scinderin(SCIN)expressed higher in GC tissues compared with adjacent normal tissues,and that the overall survival rate for patients with SCINlow was significantly higher than that of SCINhigh patients.Moreover,we have also proved that SCIN had the capability to promote invasion,metastasis and tumorigenicity of GC cells.However,the underlying mechanisms of SCIN-promoting invasion in GC cells still remained to be illustrated.This study aims to illustrate the mechanism of SCIN-promoting invasion in GC cells and to explore the possibility that SCIN be a novel prognostic marker and a potential therapeutic target for human GC.Materials and Methods:1.Immunofluorescent assay was employed to estimate the ability of filopodia formation after SCIN was knocked down in GC cell line MGC803 and primary GC cell XN0422.2.q RT-PCR analysis was employed to measure the change in the expression of Cdc42 at RNA level after SCIN was knocked down in GC cell line MGC803 and primary GC cell XN0422.3.Western blotting analysis was employed to evaluate the change in the expression of Cdc42 at protein level after SCIN was knocked down in GC cell line MGC803 and primary GC cell XN0422.4.All experiments were conducted at least three times,and the results shown are from representative experiments.Data are presented as the mean ± SD.Statistical analysis was performed using an SPSS 17.0 software(SPSS,Inc.,Chicago,IL,USA).Student's t test was used when two groups were compared.Results:1.Immunofluorescent assay showed that SCIN-knockdown markedly impaired filopodia formation in both MGC803 and XN0422 cells compared with the wild type and mock cells.2.SCIN-knockdown resulted in the down-regulation of Cdc42 expression at RNA level in both MGC803 and XN0422 cells by 50 percent compared with the wild-type and mock cells.3.Knockdown of SCIN resulted in significant decrease of Cdc42 expression at protein level in MGC803 and XN0422 cells.Moreover,compared with Cdc42 RNA,Cdc42 protein was more significantly down regulated in both SCIN knockdown GC cell types.Conclusion:1.The formation of filopodia is involved in the SCIN-promoted invasion and metastasis of GC cells.2.Suppression of SCIN in GC cells is accompanied by decreased expression of Cdc42 at both protein and RNA levels,implying that Cdc42 is involed in the SCIN-promoted filopodia formation.Part ? The establishment and characterization of a new gastric GIST cell lineBackground and objective:Gastrointestinal stromal tumors(GISTs)are the most common type of mesenchymal tumors of the gastrointestinal tract arising from interstitial cells of Cajal or their precursors.The annual incidence of GISTs is about 10 – 20 cases per 1 million individuals.GISTs commonly occur in the stomach(50–70%),followed by small intestine(20–30%)and other regions including the esophagus,colon and rectum or even sites out of the gastrointestinal tract(less than 10%).Currently,GISTs are usually treated by the resection of the primary tumor combining the consideration of targeted therapy with imatinib.The gain-of-function mutation within the c-kit(75%–85%)or PDGFRA(platelet-derived growth factor receptor alpha;5%–7%)gene is the tumor-initiating factor in most GIST cases and lead to constitutive activation of receptor tyrosine kinase,which in turn results in unlimited proliferation of GIST cells.As a result,GISTs could successfully be treated with tyrosine kinase inhibitors such as imatinib mesylate.Disappointingly,imatinib is rarely curative and about 50% patients finally acquire resistance and experience recurrence within a median time of 2 years of treatment.Although several reasons for imatinib resistance have been discovered,the underlying molecular mechanisms still remain largely unknown.However,deeper researches are hampered by a lack of proper and available GIST cell lines.To our knowledge,there are only several reported GIST cell lines including GIST-T1,GIST-H1,GK1 C and GK3 C.Our purpose is to establish a new GIST cell line with complete characteristic information and extensive availability in order to promote the study on GIST.Materials and Methods:1.Tissue block method was employed for primary cell culture.Partial passage was conducted after cancer cells migrating from the tumor tissues to concentrate GIST cells.Elimination of fibroblasts and stable GIST cells were acquired through nitrogen cryopreservation,recovery and repeated passages.2.Immunohistochemical staining was conducted to test the expression of CD117,CD34,DOG-1,?-SMA and S-100 in primary tumor tissues and xenografts.3.Immunofluorescent assay was employed to test the expression of CD117 in GIST cells.4.Immunocytochemistry was conducted to test the the expression of CD117,CD34,DOG-1,?-SMA and S-100 in GIST cells.5.The growth curve of GIST cells was plotted and by which the cell doubling time was counted.Cell microstructures were observed using transmission electron microscope and karyotype analysis was performed to evaluate the chromosome disorders.6.Xenograft assays were carried out to test the tumorigenic ability.Four groups of different numbers of GIST cells,including 1×104,1×105,5×105 and 1×106,were implanted subcutaneously into nude mouse(four nude mice for each group),respectively.7.Imatinib was used to test the drug susceptibility and the IC50 was calculated.8.Exon 8,9,11,13 and 17 of c-kit DNA and exon 12,14 and 18 of PDGFRA were sequenced and mutations were then analyzed.9.Statistical analysis was performed using an SPSS 17.0 software(SPSS,Inc.,Chicago,IL,USA).Data are presented as the mean ± SD.Student's t test was used when two groups were compared.Comparison of xenograft weight was carried out through nonparametric tests and imatinib IC50 was calculated through curve fitting analysis.Results:1.GIST cells were successfully isolated and cultured by tissue block method.Fibroblasts were eliminated from the GIST cells by nitrogen cryopreservation,recovery and repeated passages.2.Immunohistochemical analysis of the primary tumor and xenografts showed that CD117,CD34 and DOG-1 were positive,while S-100 and ?-SMA were negative.3.Immunofluorescent assay showed that CD117 was expressed in GIST cells and mainly located on the cell membranes.4.Immunocytochemical analysis of GIST cells showed that CD117,CD34 and DOG-1 were positive while ?-SMA and S-100 were negative.These results were consistent with the molecular expression patterns of the primary tumor and xenografts.5.A 24.063 h of cell doubling time for the new cell line was determined by growth curve over a culture period of five days.Transmission electron microscope revealed that the nuclei were obviously larger and the N/C ratio increased compared with normal cells.The nuclear membranes were clear and contained one or two large nucleolus.The morphology of nuclei was irregular and the microvilli were reduced or disappeared.The chromosome analysis showed that the cells were triploid with 64-68 chromosome numbers.Abnormal chromosome numbers and structures were observed in chromosome Y,1,2,3,4,6,7,9,10,11,12,13,15,16,17,19,21 and 22 including translocation,deletion and duplication.6.The new cell line had high tumorigenesis.Three weeks after injection,visible tumors developed in all mice at the site of implantation.Group of 1×106 cells developed visible tumors as early as 7 days and 1×104 cells developed visible tumors on the day 13.At the end of 22 days,all the 16 mice were sacrificed to harvest the xenografts and the tumor mass was weighed.The weight of the xenografts was positively correlated with cell number(P<0.05).7.The imatinib IC50 was 0.5343?mol/L,indicating that the cells were sensitive to imatinib.8.No mutantion was found in exon 8,9,11,13 and 17 of c-kit and exon 14 of PDGFRA.A mutation of A>G at genetic locus 1701 in exon 12 of PDGFRA and a mutation of C>T at genetic locus 2472 in exon 18 of PDGFRA were observed.However,both the mutations were synonymous with no alteration of amino-acid coding.Conclusions:1.A new GIST cell line was successfully established from a primary tumor of patient with gastric GIST.2.The new cell line exhibits CD117,CD34 and DOG-1 positive,while S-100 and ?-SMA negative,and a cell doubling time of 24.063 h.3.This new GIST cell line is tumorigenic and sensitive to imatinib with an IC50 of 0.5343?mol/L.