Antitumor Activity Of Metronomic Chemotherapy In Human Gastric Cancer Xenograft Models

BackgroudGastric carcinoma remains a common disease worldwide.It represents the fourth most frequent malignancy and second leading cause of cancer-related death worldwide.Surgical resection is the standard treatment for patients with early-stage gastric cancer.Howerver, 80 to 90%of all patients are either diagnosed at an advanced stage when the tumour is inoperable.Patients with advanced disease are mainly treated with chemotherapy. Although several randomised trials have provided evidence that chemotherapy improves survival in these patients,the standard chemotherapeutic regimens often seriously impair the quality of life and cause serious side effects,which pose serious constraints on the use of chemotherapy.In order to avoid the problems caused by traditional chemotherapeutic treatments, several researchers began to search for new modalities of drug administration oriented towards a more efficient and non-toxic antitumoral and/or antimetastic therapy. Angogenesis is necessary to sustain the growth of both the primary tumor and the development of metastases.Therapeutic targeting of tumor angiogenesis was first proposed by Folkman in 1971.From then on,the concept of angiogenesis as a therapeutic target in cancer has gained considerable momentum.Experiments had shown that some cytoxic drugs inhibited angiogenesis effectively,especially when administered more frequently and at lower dosage.Compared to maximum tolerable dose(MTD) chemotherapy,drugs being given at lower doses more frequently have stronger target effects on tumor vessel endothelial cells.This type of regimen is called metronomic chemotherapy or antiangiogenic chemotherapy.The metronomic chemotherapy affects the actively dividing and genetically stable endothelial cells,thereby reducing the chances of drug resistance to minimum.Because of its lower dosage,and more frequent schedule,it has lower toxicity. To develop a new approach for the treatment of gastric cancer,we want to explore whether antiangiogenic effect will be seen in nude mice bearing gastic cancer cells with this therapeutic approaches.Recent evidences suggest that angiogenesis may not be the only mechanism by which tumors acquire a microcirculation.Tumour cells are able to behave like stem cells. Therefore,cancer cells can mimic other cell types and can adapt to microenvironmental changes.Tumour cell plasticity refers to the ability of tumour cells to express genes associated with multiple cellular phenotypes.Vasculogenic mimicry,in which tumour cells express genes associated with endothelial cells and form fluid-conducting channels,is an example of this plasticity.Some evidences suggested that gastric carcinoma cell lines expressed a set of genes,many of which had been implicated in epithelial cell biology, such as Von Willebrand factor,CD31,VE-cadherin.That means gastric carcinoma cells may be have tumor cell plasticity,and form VM in vivo.Based on these evidences,we want to explore wethere MKN-45 tumor cells can form VM in vivo and the effect of metronomic chemotherapy on VM.ObjectTo assess endothelial progenitor cells(EPCs) in the peripheral blood of SGC 7901, MKN45,Huh-7 and U87 xenograft in BALB/c nude mice,to determined the optimum biologic dose(OBD) of metronomic LIFT,and to assess antiangiogenic effect and its effect on vasculogenic mimicry of metronomic UFT in human MKN-45 gastric cancer,compare its efficacy with MTD chemotherapy and metronomic CTX.Materials and Methods1.MKN-45 and SGC-7901 human gastric carcinoma cell lines,Huh-7 human hepatoma cell lines and U87 human glioblastoma cell lines were inoculated subcutaneously in nude mice to develop s.c.growing tumors.Nude mice were sacrificed at the 3th week after tumor implantation,EPCs in the peripheral blood of SGC-7901, MKN-45,Huh-7 and U87 xenograft in BALB/c nude mice were measured by 3-color flow cytometry.CEP(EPC in the peripheral blood) subset were depicted using the endothelial murine markers VEGF receptor 2 fetal liver kinase 1(flk-1),Sca-1,and CD117.After nude mice bearing MKN-45 human gastric cancer cell lines were treated with 0,5,10,15,20, 25mg/kg/d UFT,mice were sacrificed at 3th week,evaluation of CEPs was carried out on blood by flow cytometry.2.Except for blank group,which was tumor-free nude mice,xenografts of MKN-45 gastric cancer cell lines were subjected to either continuous treatment with normal saline, low doses of UFT(20mg/kg/d),maximum tolerable doses of UFT(50mg/kg,5 consecutive days) and low doses of cyclophosphamide(CTX,20mg/kg/d).At the 1th,2th,3th week after the treatment,5 in each group were sacrificed,and evaluation of CEPs was carried out on blood by flow cytometry.Tumor microvessel density was evaluated by immunohistochemistry at the 3th week after the treatment.3.Nude mice bearing MKN-45 human gastric carcinoma cell lines were deviding into three group:control group(A),UFT LMD(20mg/kg/d) group(B),UFT MTD (50mg/kg,5 consecutive days) group(C).At the 1th,2th,3th week after the treatment with normal saline or UFT,5 in each group were sacrificed.The morphological characteristics of slides of tumor specimens stained with H&E and PAS were examined in microscope, and counted the numbers of vasculogenic mimicry(VM).Immunohistochemical staining was used to analyze the expression of CD31.At 3th week after the treatment,the mouse sera were collected and measured for human VEGF by ELISA.Results1.In immunodeficient mice bearing MKN-45 and SGC 7901 human gastric carcinoma cell lines,Huh-7 human hepatoma cell lines and U87 human glioblastoma cell lines,increases in the levels of CEPs were observed.Nude mice bearing U87 human glioblastoma cell lines expressed the highest numbers of CEPs.In contrast,nude mice bearing Huh-7 human hepatoma cell lines were found to have much lowest numbers of CEPs.Moreover,we also observed a correlation between the number of CEPs and cancer cell lines.Using CEPs as a biomarker,we determined that the OBD of metronomic UFT administered daily was 20mg/kg.2.In immunodeficient mice bearing MKN-45 human gastric carcinoma cell lines, metronomic UFT inhibited the mobilization of CEPs and reduced MVD,but metronomic CTX didn't,although in group CTX LMD,inhibiton rate of tumor was higher than in group UFT LMD.We observed a correlation between the number of CEPs and MVD.3.MKN-45 tumor cells were ability to form VM in vivo.We found metronomic UFT inhibited the VM formations by MKN-45 gastric cancer cells,but MTD UFT didn't.Conclusion1.There are different levels of CEPs in the nude mice bearing different cancer cells.2.We determine the OBD of metronomic UFT by detection of CEPs was 20mg/kg.3.Metronomic UFT has antiangiogenic effect in human MKN-45 gastric cancer.4.Metronomic UFT can inhibited VM formations by MKN-45 gastric cancer cells.