A Study On The Antitumor Activities In Vivo And In Vitro Of Lung Cancer Dendritic Cell Vaccine Modified By Mutant K-ras Genes

Lung cancer is one of the most malignant tumors which is hazarding to the people's health and the incidence is increasing over the past few decades in the world, but its prognosis is very poor, only 10-15% patients are cured. Current therapy of lung cancer is multimodality therapy including surgical treatment, chemotherapy and radiotherapy.Most patients, however, lose the chance of operation when they are diagnosed to have lung cancer. Many patients who underwent complete resection of the cancer will develop recurrent or metastatic disease, and most will die as a consequence. Although chemotherapy and radiotherapy can kill tumor cell, they can hurt the normal cell, too. DC-based vaccination represents a potentially powerful strategy for cancer biotherapy. In this study, recombinant adenovirus of mutant human K-ras/V12 and control adenovirus are constructed by the modified homologous recombination in bacteria. 3LL-pcDNA3-K-ras/V12 lung cancer cell line which can express mutant human K-ras/V12 gene was constructedby the lipofectin-mediated gene transfection method. Then, Dendritic cell (DC) from mice bone marrow were transfected with the recombinant adenovirus of mutant human K-ras/V12. The antitumor effect and its mechanisms of the dendritic cell modified by mutant K-ras/V12 was determined in vivo and in vitro.The results showed that:l.Over 25% positive recombinant bacterial clones was obtained after co-transformation of BJ5183 bacterial cells with pAdTrackCMV-K-ras/V12 and pAdEasy-1 by the method of CaCl₂.2.PCR and DNA sequencing indicated each the recombinant adenoviruscontained the insert of K-ras/V12.The titre of purified Ad was 1.2 × 10¹²pfu/ml, and 70% Dendretic cells(DCs) were infected with Ad-K-ras/V12 when MOI reached 100.3.Lewis lung cancer cell line 3LL were transfected with a pcDNA3-K-ras/V12 plasmid DNA using the lipofectin-mediated gene transfection method. Using G418 screening them,the cell transfected pcDNA3-K-ras /12(Val) and the cell transfected pcDNA3 were survived,but the lewis lung cancer cell line 3LL can not survive. 3LL-pcDNA3-K-ras/V12 cell line was half adhensive cell. The shape of the transfected cells have no significant difference with the 3LL-pcDNA3 cells and 3LL cells.4. It was confirmed that the K-ras/V12 gene had been transfected into 3LL-pcDNA3-K-ras/V12 cells using PCR methods.5. The results of FACS showed that the expression level of p21, ERK, VEGF, CD44, MMP in 3LL-pcDNA3-K-ras/V12 cells was remarkly higher than that in 3LL-pcDNA3 and the 3LL cells (P<0.05 or P<0.01) .6. The 3LL lung cancer cell line transfected with mutant K-ras gene cansurvive by logarithem after culture. The doubling time was 18.5hrs.7.The sofe agar colony forming rate of the 3LL transfected with mutant K-ras gene was 27%. Comparing with the 3LL transfected with vector and the 3LL ,it had no significant difference(/)>0.05).8.The 3LL-pcDNA3-K-ras/V12, 3LL-pcDNA3 and 3LL lung cancer cell line all had the bility of tumorigenesis in C57BL/6 mice. The average tumor weight was 3.80 ± 0.3 lg in 3LL-pcDNA3-K-ras/V12 lung cancer cell line ,4.05±0.33g in 3LL-pcDNA3 lung cancer cell line and 3.91±0.10g in 3LL lung cancer cell line.No significant difference was observed among the three groups (P>0.05).9.The mice bone marrow DC(BmDC) grew as typical DC which cytoplasm is protrusion like polythoorm by using rmGM-CSF and rmIL-4,the harvest rate of DC were 81% > 96% > 86.65% respectively on the 3th ,5th ,7th post-culture days.BmDC highly expressed B7-1 (77%) ,B7-2 (80%) ,MHC II (68%) ,CDllc(69%),CD40(70%)andCD54 (84%) byFACS.10.The results of FACS showed that no significant difference of molecular expression was observed between the DC transfected with adenovirus and DC without transfected.11.It was confirmed that the K-ras/V12 gene had been transfected into the DC-K-ras/V12 cells using PCR method.12.The lymphocytes proliferation stimulated by DC modified with mutant K-ras gene was significantly higher than that modified with Ad-c or not modified DC (PO.05). When DC:T=1:5, the effect is more markly (P<0.0\).13.DC vaccine modified by mutant K-ras gene could greatly inducedCTL activity against 3LL-pcDNA3-K-ras/V12 lewis lung cancer cell line that transfected with the same mutant K-ras gene.but it also could induce certain CTL activity against 3LL-pcDNA3 lewis lung cancer cell line transfected with vector and 3LL lewis lung cancer cell line. But it have no cytotoxicity against B16. The CTL activity against 3LL-pcDNA3-K-ras/V12 lung cancer cell line is much stronger than that against 3LL-pcDNA3 and 3LL lung cancer cell lines (/><0.05).The spleen lymphocytes from the mice vaccined with control, vector and non-modified DC groups did not induce specific cytotoxicity against 3LL-pcDNA3-K-ras/V^ 3LL-pcDNA3 and 3LL lung cancer cell lines.14.The tumorigenesis of transplanted tumor with 3LL-pcDNA3-K-ras/V12,3LL-pcDNA3 and 3LL lung cancer cell lines in the mice vaccined with mutant K-ras gene modified DC were significantly suppressed.The volume and weight of transplant tumor in the DC-K-ras/V12 group was remarkably lower than those in controK vector and non-modified DC groups(P<0.05).The inhibitory rate of tumor growth in the DC-K-ras/V12 group was significantly higher than that in the control -, vector and non-modified DC groups(P<0.05). However the volume and weight of transplant tumor ,and inhibitory rate in the control,vector and non-modified DC groups had no significant difference(jP>0.05).15.Comparing the tumorigenesis of transplanted tumor of 3LL, 3LL-pcDNA3 and 3LL-pcDNA3-K-ras/V12 lung cancer cell lines in the mice vaccined with mutant K-ras-modified DC, it was showed that suppression of tumorigenesis induced by DC vaccine modified by mutant K-ras gene in 3LL-pcDNA3-K-ras/V12 was remarkly higher than in 3LL and3LL-pcDNA3(P<0.01). The volume and weight of transplant tumor of 3LL-pcDNA3-K-ras/V12 lung cancer cell line was remarkly lower than those of 3LL-pcDNA3 and 3LL lung cancer cell lines, and the inhibitory rate was significantly higher than those of 3LL-pcDNA3 and 3LL lung cancer cell lines CP<0.01).16.The lung metastastic rate of transplanted tumor of 3LL-pcDNA3-K-ras/V 12 lung cancer cell line in the mice vaccined with mutant K-ras gene modified DC(30%) was remarkably lower than those in the control(90%), vector(80%) and non-modified DC groups(80%)(P<0.01). The lung metastatic rate of transplanted tumor of 3LL-pcDNA3 lung cancer cell line in the mice vaccined with mutant K-ras gene modified DC (50%) was also remarkably lower than those in the control (80%), vector(80%) and non-modified DC groups(70%XP<0.01) The lung metastatic rate of transplanted tumor of 3LL lung cancer cell line in the mice vaccined with mutant K-ras gene modified DC (40%) was remarkably lower than those in the control(80%),vector(60%) and non-modified DC groups (70%), too17.Comparing lung metastatic rate and the number of lung metastatic loci of transplant tumor of 3LL-pcDNA3-K-ras/V12, 3LL-pcDNA3 and 3LL lung cancer cell line,the lung metastatic rate and metastatic loce in the DC-K-ras/V12 group was significantly lower than those in the control, vector and non-modified DC groups(jP<0.01).Conclusion: (1) The modified method of homologous recombinantion in bacteria is a convenient and high efficient method to prepare recombinant adenovirus and the pepared Ad-K-ras/V12 can effectively mediate target geneexpression in infected cells.(2)3LL-pcDNA3-K-ras/V12 lung cancer cell line which can express mutant human K-ras/V12 gene was constructed by the lipofectin-mediated gene transfection method .3LL-pcDNA3-K-ras/V12 lung cancer cell line had higher expression level of p21, MMP, ERK, VEGF and CD44 genes comparing with 3LL and 3LL-pcDNA3 lung cancer cell lines. (3) Specific antitumor immune response could be induced effectively by vaccination with mutant K-ras gene modified DC.The DC vaccine modified by mutant K-ras gene can remarkably suppress the growth and distant metastasis of 3LL-pcDNA3-K-ras/V12 that can express the same mutant K-ras gene; (4) The DC modified with mutant K-ras gene may be promising tool for the immunotherapy of lung cancer. It can provide a new strategy for lung cancer.