Effect Of Recombinant Adenovirus-p53 On Growth And Chemosensitivity, Radiosensitivity Of Human Gastric And Colonic Carcinoma Cells With Thermosensitivity

Objective:1.To evaluate the effect of adenovirus-mediated p53 gene (Adp53) on apoptosis and chemosensitivity,thermosensitivity, radiosensitivity of human gastric carcinoma cell lines.2.To investigate the Change of the expression of p53,bc1-2,and bax after hyperthermia, chemotherapy and radiotherapy in human colon cancer transplanted in nude mice.Methods:Recombinant adenoviru s mediated p 53 gene(rAd-p53) was transfected in to human gastric cancer cells with unknown p53 genetic status.P53 protein expression was detected with Western blot assay.Cell survival and apoptosis of the gastric cancer cells was assessed with MTT assay.Cell cycle distribu tion and apoptotic rate were determined with flow cytometry.Nude mice xenograft models of W and M cell were intratumorally injected with Adp53,48h later,and after the heating at 43℃for 0.5h,relative volume growth curve of the tumor was depicted to evaluate the tumor regression.Nude mice xenograft models of W and M cell were intratumorally injected with Adp53 and 48 h later were irradiated with 6 Gy.Relative volume in growth curve of tumor was used to observe tumor regression.Human colon cancer cells(HT29) were transplanted into the behind limbs of the naked mice.Under the laboratory simulated conditions of hyperthermia(43℃,60min),referring to the clinical chemotherapy prescription for human colon cancer(MMC),the actual doses were calculated,and referring to the clinical radiotherapy prescription for human colon cancer,the actual radio doses were calculated.In this experiment,the mice were divided into 6 groups: hyperthermia group,chemotherapy group,radiotherapy group, chemohyperthermia group,radiohyperthermia group,and radiochemohyperthermia group.The mice were sacrificed and the tumor tissue was taken at different time points(before treatment,at 2h,4h, 8h,12h,24h,48h,and 72h after treatment).The morphologic changes in tumor cell memberance,plasm and nucleus evoked by the expression P53, Bc1-2,and Bax were observed by the method of immunohistochemistry stain(S-P method).Results:Inducing of G2/M arrest and apoptosis and inhibition of tumor cell pro liferation were observed after the infection of Adp53,and efficiently P53 protein expression was demonstrated in the human gastric cancer cells in this study.A denovirus mediated p53 enhanced the sensitivity of gastric cancer cells to cisplat in w ith the dependence of time and dose.Thermo enhancement ratio of Adp53 at 43℃for 0.5h was 1.7 for W cell planting tumor and 1.6 for M cell planting tumor in vivo.The antitumor enhencement ratio of Adp53 at 6 Gy was 1141 for W cell2implanted tumor and 1191 for M cell2implanted tumor in vivo. All of the six treatment methods can reduce the expression of P53,bc1-2 and raise the express of bax in various degree;The allied therapy of hyperthermia,chemotherapy,and/or radiotherapy made this upregulation more prominent than any single therapy.Conclusion:Adp53 transfer increases the cellular apoptosis and sensitivity of human gastric cancer cells in vitro,and the combination of p53 genetherapy with chemotherapy may enhance the adenovirus mediated p53 on chemosensitivity,thermosensitivity,radiosensitivity of gastric cancer cells.Hyperthermia makes as an intensifier to chemotherapy and radiotherapy probably by enhancing chemotherapy and radiotherapy' s ability of changing the expressiong of apoptosis genes in tumors.