Experimental Studies On The Anti-tumor Effects Of PEgr-IFNγ-Endostatin Gene-radiotherapy And Its Mechanism

Radiotherapy is one of the most important methods for the treatment of tumors, butits application is currently limited by the side-effects such as the radiation-induceddamages in the normal tissues nearby and the radiation-resistance of certain tumors. Tosolve these problems we need to find an effective way which could decrease the dose ofradiation and at the same time increase the suppression of tumors. Gene-radiotherapy, thecombination of gene therapy and radiation therapy , is a new paradigm for cancertreatment and offers great potential for the treatment of localized human cancers. Muchattention have focus on the p53-independent radiation-inducible Egr-1(early growthresponse-1 ) promoter for anti-tumor gene-radiotherapy. It has been repeatedly reportedthat ionizing radiation can activate Egr-1 gene promoter and consequently induce theexpression of downstream single gene. For further enhanceing anti-tumor effects ofgene-radiotherapy combined gene-radiotherapy is particular promising. In this study,according to the mechanism of activation of transcription of the Egr-1 gene induced byradiation, the direct and indirect anti-tumor effects of IFN-γand the anti-angiogenesiseffects of Endostatin, we construct a radiation-inducible expression vector pEgr-IFNγ-Endostatin that simultaneously expresses IFNγand Endostatin genes, Compare theanti-tumor effects in vivo of pEgr-IFNγ-Endostatin gene-radiotherapy with that ofpEgr-IFNγand pEgr-Endostatin gene-radiotherapy and study its anti-tumor mechanism.This study represents a novel strategy for effective gene-radiotherapy of cancer and mighthave wide application in the future.1 Construction of recombinant plasmidsRecombinant plasmids pEgr-IFNγ, pEgr-Endostatin and pEgr-IFNγ-Endostatinwere constructed successfully with gene recombinant technique after identification withrestriction enzyme.2 Expression of IFNγand Endostatin in Lewis lung carcinoma cells induced by radiation in vitro2.1 Expression of IFNγand Endostatin in LLC cells transfected by pEgr-IFNγ-Endostatin after different doses X-ray irradiationRecombinant plasmid pEgr-IFNγ-Endostatin were transferred into Lewis lungcarcinoma cells by liposome in vitro. The correlation of dose and effect of the expressionof IFNr and Endostatin genes induced by X-ray were detected by ELISA. After differentdoses of X-ray irradiation, the expression of IFNr and Endostatin in the supernatant ofLewis lung carcinoma cells were significantly higher than that in 0 Gy group. After 5 GyX-ray irradiation, the expression of IFNr and Endostatin reached the highest and were4.14 and 2.92 times high than in 0 Gy group respectively.2.2 Expression of IFN γand Endostatin in LLC cells transfected bypEgr-IFN-Endostatin at different time after 2.0 Gy X-irradiation Recombinant plasmid pEgr-IFNγ-Endostatin were transferred into Lewis lungcarcinoma cells by liposome in vitro. The time-course rules of the expression of IFNr andEndostatin genes induced by X-ray were detected by ELISA.The content of IFNr andEndostatin in supernatant increased after 2 Gy X-ray irradiation and were 3.75 and 3.02times as much as that in 0 Gy group respectively 36 h after irradiation.3 Anti-tumor effects of pEgr-IFNγ-Endostatin gene-radiotherapy in vivo3.1 Effects of pEgr-IFN-Endostatin gene-radiotherapy for only once on tumor growth ofLLC cells implanted in C57BL/6J mice Recombinant plasmids packed by liposome were injected locally into the tumors ofthe mice, and then the tumors were irradiated with 5 Gy X-ray. The tumor growth rate ofthe mice in dule-gene-radiotherapy group were significantly lower than that of controlgroup(P<0.01~0.001), 5 Gy group(P<0.01~0.001) and single-gene-radiotherapygroup(P<0.05~0.001) 6~18 d after gene-radiotherapy, and the mean survival period ofwhich were longer(P<0.05~0.001). In addition, the number of lung metastatic nodes andthe tumor weight of the mice in dule-gene-radiotherapy group were significantly lowerthan that of other groups 18 d after gene-radiotherapy(P<0.01~0.001, P<0.01~0.001).The tumor weight of the mice in dule-gene-radiotherapy group were only 18% of that ofthe mice in control group. The results showed that the anti-tumor effects in vivo ofpEgr-IFNγ-Endostatin gene-radiotherapy were better than that of pEgr-IFNγor pEgr-Endostatin gene-radiotherapy with the tumor growth rate, number of lung metastaticnodes and tumor weight decreased and mean survival period prolonged.3.2 Effects of repeated pEgr-IFN-Endostatin gene-radiotherapy on tumor growth of LLCcells implanted in C57BL/6J mice Recombinant plasmids pEgr-IFN-Endostatin packed by liposome were injectedlocally into the tumors of the mice for four times every other day each followed by 2.5 GyX-ray irradiation. The tumor growth rate of the mice in dule-gene-radiotherapy for fourtimes group were significantly lower than that of dule-gene-radiotherapy for only oncegroup(P<0.01~0.001) 12~18 d after gene-radiotherapy. The tumor growth rate of themice in dule-gene-radiotherapy for four times group were only 9.6% of that of the mice incontrol group and 32% in dule-gene-radiotherapy for only once group 18 d aftergene-radiotherapy. The mean survival period of the mice in dule-gene-radiotherapy forfour times group were significantly longer than that of the mice in other groups(P<0.001). Compared with control group and dule-gene-radiotherapy for only once group,The mean survival period of the mice in dule-gene-radiotherapy for four times group wereprolonged for 42 d and 11 d respectively. In addition, the number of lung metastatic nodesand the tumor weight of the mice in dule-gene-radiotherapy for four times group weresignificantly lower than that of dule-gene-radiotherapy for only once group 18 d aftergene-radiotherapy ( P < 0.001, P < 0.01 ) . The tumor weight of the mice indule-gene-radiotherapy group were only 6% of that of the mice in control group. Theresults showed that the anti-tumor effects in vivo of repeated pEgr-IFNγ-Endostatingene-radiotherapy were better than that of pEgr-IFNγ-Endostatin gene-radiotherapy foronly once.4 Mechanism of anti-tumor effects of pEgr-IFNγ-Endostatin gene- radiotherapy in vivo4.1 Immunological mechanism of anti-tumor effects of pEgr-IFNγ-Endostatin gene-radiotherapy in vivo C57BL/6J mice-bearing Lewis lung carcinoma cells were divided randomly into sixgroups: control, 5 Gy alone, pIRES1neo combined with 5 Gy, pEgr-IFNγcombinedwith 5 Gy, pEgr -Endostatin combined with 5 Gy, pEgr-IFNγ-Endostatin combined with5 Gy group. Cytotoxic activity of splenic CTL, NK and TNFαsecretion activity ofperitoneal macrophages of the mice in different groups were examined 15 d aftergene-radiotherapy. Cytotoxic activity of splenic CTL, NK and TNFαsecretion activity ofperitoneal macrophages of the mice in pEgr-IFN γ-Endo and pEgr-IFN γgene-radiotherapy group were significantly higher than those in control group, 5 GyX-ray irradiation group and pEgr-Endo gene-radiotherapy group(P<0.01~0.001). Thereis no significant difference between the mice in pEgr-IFNγ-Endo and pEgr-IFNγgene-radiotherapy group.4.2 Expression of IFNγand Endostatin in cytoplasm of LLC cells implanted inC57BL/6J mice 1 day after gene-radiotherapy Experimental groups were the same as mentioned above. The concentration of IFNγand Endostatin in cytoplasm of LLC cells were detected with ELISA assay. No IFNγand Endostatin protein were detected in LLC cells of the mice in control, 5 Gy alone andpIRES1neo combined with 5 Gy group. However, IFNγor Endostatin protein waspresent in LLC cells treated with gene-radiotherapy group. The concentration of IFNγ...