Targeted Gene Therapy Of HSV-TK/hIL12 Fusion Gene Controlled By HSLPI Gene Promoter For HNSCLC In Vitro

The incidence and the mortality of lung cancer remain high and on theleading increasing in all tumors not only in china but also in other countries.More than 80% of the histological types of lung cancer is non–small cell lungcancer(hNSCLC), and most of the patients were often found in advanced orterminal stage when seeing doctor. Conventional treatments, such as surgery,radiotherapy and chemotherapy, are not effective enough to overcome it.Recently, herpes simplex virus-thymidine kinase with its prodrugganciclovir(HSV-TK/GCV), as one of approaches to gene therapy for cancer,has been proven to be more effective cure for tumor. It is one of the mostpromising development of therapeutic methods. At present, targeted andcombined gene therapy are two key points in this field.Some researches have showed that HSV-TK gene combined withimmunity gene had resulted in cooperative and more effective antitumoreffects. On the one hand , TK/GCV may directedly kill some tumor cells torelieve the burden of it and indirectedly kill neighbour or metastasistumor cells through local or distant bystander effects as well. On theother hand, the administration of immunity gene may activate the body'santitumor immunity function and enlarge the bystander effect of thekilling of TK/GCV, so that it could reduce the recurrence of tumor anderadicate the tumor to improve the antitumor effects. Interleukin12(IL-12)is one of the immunity factors, and its importance in the tumorimmunotherapy has been widely demonstrated for its potent and variousantitumor activity from the strong immunity functions of its regulating body'sacquired immunity as well as innate immunity. Some researchers reported thatthe combining gene therapy with adenoviral vector-mediated HSV-TK andmIL-12 gene for mouse prostate cancer resulted in significant therapeuticactivities in vitro and vivo assays. However, the majority of reports on thecombining genes of HSV-TK and IL-12 only located in the application ofmIL-12 gene. As to hIL-12, their cooperative antitumor effect remainsobscure.It's a novel important target strategy to modulate the specific expressionof HSV-TK in tumor cells with tumor-specific promoter. Now, it is found tohave more than ten sorts of lung cancer-specific promoter. Despite of havingshowed anti-lung-cancer-specific activities in some studies, manydisadvantages still remains. So it is more necessary to explore a novel lungcancer-specific promoter for improving the target strategy in gene therapy oflung cancer.Recently, some studies have showed that human secretoryleukoprotease inhibitor (hSLPI) gene is expressed at high levels insome human carcinomas, including lung, ovarian and cervix, butminimally in normal tissue such as liver, endocrine glands and bloodsystem. Studies have been performed employing hSLPI promoter todrive therapeutic gene expression in cervical or ovarian cancer andhave showed an effective and specific antitumor activity. In 2004,Maemondo et al utilized this promoter to control replication-competentadenovirus for a specific gene therapy of hNSCLC including squamous cellcarcinoma (HS-24) and adenocarcinoma (A549 and H358), which resulted inspecific anti-lung-cancer. Comparing to the promoters which target to someone histology of lung cancer, hSLPI promoter could target more various sortsof lung cancer such as squamous cell carcinoma and adenocarcinoma.However, the related reports of this promoter mainly explored its selectivegene expression in an adenoviral vector. It is not an appropriate approach forclinic application. Some studies also suggested that some promoters losed theirfidelity when placed in an adenovirus and becomed either less efficient or lessspecific. Now it is still unknown that whether the selecting expression and thetargeting antitumor activity controlled by this promoter in a non-adenoviralvector are influenced or not.As a result, there are two aims in this study. One is selecting hSLPIpromoter as the switch on gene therapy for hNSCLC. After the basicexperiments of cloning hSLPI promoter from the genomic DNAs of humanperipheral blood by PCR and constructing objective gene expression vectorscontrolled by such promoter in series, it will be investigated that if the hSLPIpromoter could modulate selective gene expression in the non-small cell lungcancer cell lines. The other will further explore the targeting and cooperativeanti-lung-cancer activities with HSV-TK/hIL12 fusion gene controlled byhSLPI promoter in vitro. Therefore, the experimental foundation and evidencewill be provided for further studies in vivo on targeted gene therapy ofhNSCLC under the control of this promoter, and a novel molecular targetingapproach will be offered for the treatment of lung cancer. The followingresults have been obtained in this study:1. The construction of EGFP eukaryotic expression vector under thecontrol of hSLPI promoterhSLPI promoter was cloned from the genomic DNAs of humanperipheral blood by PCR and was sequenced,which demonstrated that thesequence of it was in accordance with that of the 5′flanking transcriptionalregulating region of hSLPI gene. Later the digested and purified hSLPIpromoter was ligated into the linear vector pcDNA3.1(+) which was digestedby the same endonuclease, and so did the EGFP gene fragment digested fromplasmid pEGFP. Then the recombinant eukaryotic expression vector,phSLPI-EGFP,further identified by restriction endonuclease was successfullyconstructed. It provided the experimental foundation for further employing thispromoter-controlled selective expression of therapeutic genes in the non-smallcell lung cancer cell lines.2. The selective expression of phSLPI-EGFP in tumor cell linesphSLPI-EGFP was tansfected into four tumour cell lines, Hela, A549,SPC-A1 and HepG2, by lipofectamine, and the stablely-transfectant cloneswere established through exposure to G418. Then it showed that theexpression of EGFP protein observed under fluorescence microscope wasunselective in all four cell lines transfected with pEGFP, while selective inHela, A549 and SPC-A1, not in HepG2 which were transfected withphSLPI-EGFP. The results indicated that the cloned hSLPI promoter coulddrive the following gene selectively expressing in hNSCLC cell lines. Itoffered the experimental evidence for further exploring the targeted genetherapy for lung carcinoma with the regulation of hSLPI promoter.3. The construction of HSV-TK or HSV-TK/hIL12 gene eukaryoticexpression vectors under the control of hSLPI or CMV promoterDNA sequencing demonstrated that the cloned sequences of hIL-12,HSV-TK and HSV-TK-IRES were in accordance with those of the conunterpart of the plasmid pGT60-hIL-12. Then the recombinant eukaryoticexpression vectors, pcDNA3.1-TK, pcDNA3.1-TK/hIL12, phSLPI-TK andphSLPI-TK/hIL12, identified by restriction endonuclease and PCR weresuccessfully constructed. It provided the basic experimental conditions forserial analysis of selective expression of HSV-TK/hIL12 fusion genecontrolled by hSLPI promoter in hNSCLC cell lines and of cooperative genetherapy targetedly for lung carcinoma.4. The selective expression of therapeutic genes controlled by hSLPIpromoter in tumor cell lineshIL-12 and HSV-TK mRNA expression levels of A549, SPC-A1 and HepG2transfected stablely were determined by semiquantitative RT-PCR, and hIL-12protein expression levels and its function seperately determined by ELISA andlymphocyte proliferation assay. It showed that the expression activities ofhIL-12 gene and HSV-TK gene were unselective in all three cell linescontrolled by the CMV promoter, while selective in A549 and SPC-A1, not inHepG2 by the hSLPI promoter. The expression levels of two genes controlledby CMV promoter were higher than those controlled by hSLPIpromoter(P<0.01). The transcriptional activities of hSLPI promoter in A549and SPC-A1 were 16.89% and 20.28%, respectively, in comparation to CMVpromoter. Furthermore the expressing hIL-12 protein had proliferation activityto lymphocyte (P<0.01).5. HSV-TK gene controlled by hSLPI promoter induces hNSCLC cell linestargeted killings in vitroMTT method was applied to investigate the targeted antitumor effects invitro with HSV-TK/GCV controlled by hSLPI promoter. The results suggestedthat the killing activities of HSV-TK/GCV were non-specific in all three celllines controlled by CMV promoter, while specific in A549,SPC-A1 and not inHepG2 controlled by hSLPI promoter. Their killing activities were improvedby the increasing expression of HSV-TK gene and the increasing addition ofGCV(p<0.01). It confirmed that apoptosis exsisted in the killed cells bymicroscography and flow cytometry methods(p<0.01). Also by MTT methodthe bystander effects of killing were observed in all three cell lines even beingmixed with 10% of cells transfected with TK gene (p<0.01).6. HSV-TK/hIL12 fusion gene controlled by hSLPI promoter induceshNSCLC cell lines killings specific and cooperative in vitroBy MTT method, it showed that the specific killing activities ofHSV-TK/hIL12 fusion gene were stronger than that of HSV-TK gene(p<0.01).The increased cooperative killing activities were composed of not only theenhanced directive killing effects but also the enhanced bystander effects ofthe killing, which were produced by the coexpressing hIL-12.Taken together, we selected hSLPI promoter as the switch on gene therapyfor hNSCL. After the basic experiments of the clone of the hSLPI promoter, theconstruction of HSV-TK gene or fused with hIL-12 gene eukaryotic expressionvectors controlled by CMV or hSLPI promoter, and the stable transfection ofthose vectors into the three cell lines in series, we further confirmed that hSLPIpromoter could modulate HSV-TK/hIL12 fusion gene selective expression inthe hNSCLC cell lines and targetedly cooperative killing of those cell lines invitro. Therefore, the experimental foundation and evidence were provided forfurther studies in vivo on targeted gene therapy of therapeutic genes for lungcancer under the control of this promoter, and a novel molecular targetingapproach was offered for the treatment of lung cancer.