Synergistic Anti-tumor Effect Of Egr-hTERTC27and5-FU On Nasopharyngeal Carcinoma And Inhibition Of TCF3on Mouse Embryonic Carcinoma Growth

1Synergistic Anti-tumor Effect of Egr-hTERTC27and5-FU on Nasopharyngeal CarcinomaBackgroundNasopharyngeal carcinoma (NPC) is one of the most malignant neoplasms in thenasopharynx, especially in Southeast of China such as Guangdong, Guangxi and Fujian. Ittakes place in people of variable ages, mostly in middle-aged, but also in teenagers. NPC ishighly malignant, always with regional lymph node metastasis or distant metastasis. Thecauses for NPC are very complicated, including genetics, EB virus infection andenvironmental risk factors. Nowadays, the treatment for NPC mainly includes localradiotherapy and chemotherapy. However, there isn't any effective method to treat patientswith advanced-stage NPC and patients with early surgical operation but still with poorprognosis. Therefore, it is very important for us to seek novel strategy for NPC treatment.5-fluorouracil (5-FU), the most widely used anticancer agent, has been applied widelyto the treatment of NPC.5-FU is one kind of derivative of Uralic in which the5'-hydrogen issubstituted with fluorine.5-FU can incorporate into DNA or RNA (via UTP), leading toDNA strand breakage and a decrease in protein synthesis. However,5-FU treatment only haslimited benefit to the NPC patients because of severe side effects caused by high dosage andfailing preventing recurrence and metastasis due to limited sensitivity. Therefore, during thepast years, several strategies of gene therapy to enhance the anti-tumor effect of5-FU havebeen intensively explored to treat NPC.The early growth response protein-1(Egr-1) promoter includes6reactive elements ofCArG[CC(A+T-rich)6GG], which is sensitive to reactive oxygen intermediates. The Egr-1promoter is commonly used as a bridge in gene therapy because it can be activated by ROIs(reactive oxygen intermediates, ROIs)generated by radiotherapy or chemotherapy such as5-FU and promotes the high expression of downstream genes. Scientists proposed agene-chemotherapy hypothesis that using Egr-1promoter as a bridge to combinechemotherapy and tumor killer genes or immune therapeutic genes to achieve double anti-tumor effects, increasing the therapeutic effects and decreasing the normal tissuedamages and side effects caused by high dosage. Nowadays, the combinational treatment ofanti-tumor genes such as CD, TK, CDglyTK, Endostatin and chemotherapy using Egr-1promoter as a bridge for malignant tumors including gastrointestinal cancer, neurogliomaand liver cancer has obtained remarkable achievements.Telomerase reverse transcriptase (TERT), one catalytic protein unit of telomerase, isvery critical for maintaining the telomere length and prolonging cell life. TERT is highlyexpressed in the majority of tumor cells, but seldom in somantic cells. Therefore, TERT is anideal tumor diagnostic marker and cancer gene therapy target. hTERTC27, a27kDaC-terminal polypeptide of hTERT, is newly constructed by Prof. Lin MC of HongkongUniversity. In this polypeptide, most of the conserved hTR-binding domains and the reversetranscriptase motifs were deleted. Previously, we have reported that a27kDa C-terminalpolypeptide of hTERT (hTERTC27) is capable of inducing telomere dysfunctions, andoverexpression of hTERTC27in human cervical carcinoma HeLa cells slows down the cellproliferation and inhibits the tumorigenicity by significantly increasing anaphasechromosome end-to-end fusion. Ectopic overexpression of this polypeptide also makes theHeLa cells more sensitive to H2O2-induced oxidative injure without influencing theendogenous telomerase activity. Furthermore, hTERTC27delivered by recombinantadeno-associated virus or recombinant adeno-associated virus and adenovirus cocktailsystem suppresses tumor growth in vivo in human U87-MG glioblastoma xenograft model.Therefore, hTERTC27as a newly constructed anti-tumor polypeptide has promisingapplication prospects. However, whether hTERTC27has anti-tumor effect on NPC C666-1cells has not been investigated. Nowadays there is not any study on this issue at home andabroad.In this study, hTERTC27cDNA was constructed to the downstream of the Egr-1promoter, which was transfected into the NPC C666-1cells and induced by5-FU.5-FU cansuccessfully activate the Egr-1promoter and increase the expression of hTERTC27. Thesynergistic anti-tumor effects and possible mechanisms of5-FU and hTERTC27polypeptidedriven by5-FU activated Egr-1promoter were assayed for the first time. These resultssuggest that the synergistic effect of5-FU and Egr-1promoter driven hTERTC27might be apotential clinical strategy for cancer treatment.MethodsThe stable cell lines were established by Lipofectamine transfection method. The inducible effects of5-FU on Egr-1promoter were detected by fluorescence microscopy,flowcytometry and Western blot analysis. The in vitro synergistic anti-tumor effects of5-FUand Egr-1promoter driven hTERTC27on NPC C666-1cells were observed by MTT assay,colony formation assay, flowcytometry assay after Annexin V/PI double staining, and Tunelassay. The transplant tumor model was established subcutaneously using stable transfectedcell lines. The in vivo synergistic anti-tumor effects were observed by tumor growth curve,tumor weight and HE staining. The protein expressions of cleaved PARP,caspase-3,caspase-9and Bcl-2were detected by Western blot analysis.ResultsThe results showed that hTERTC27expression was significantly increased up to7foldsby5-FU activated Egr-1promoter in NPC C666-1cells. Over expressed hTERTC27madethe cells more sensitive to5-FU, and additionally inhibited cell proliferation about20%.Combinational therapy of over-expressed hTERTC27driven by5-FU activated Egr-1promoter (Egr-hTERTC27) and5-FU synergistically inhibited cell proliferation andpromoted apoptosis of C666-1cells for about4.8folds and1.8folds in comparison with soletherapy of hTERTC27or5-FU in vitro. In vivo experiments showed that Egr-hTERTC27and5-FU synergistically reduced tumor volume, tumor weight and local infiltration, whichmay be relative to tumor cell apoptosis. In addition, the expression of activated PARP,caspase-3and caspase-9increased significantly and the expression of anti-apoptosis proteinBcl-2decreased obviously in combinational group.ConclusionsCombinational therapy of over-expressed hTERTC27driven by5-FU activated Egr-1promoter (Egr-hTERTC27) and5-FU synergistically inhibited NPC C666-1cellproliferation and promoted apoptosis, which suggest that combinational therapy of overexpressed hTERTC27, which is driven by5-FU activated Egr-1promoter, and5-FU mayprovide a novel approach to treat cancer. 2Inhibition of TCF3on Mouse Embryonic Carcinoma GrowthBackgroundEmbryonic stem cells(ES), deviating from the inner cell mass of blastocyst, are highly undifferentiated stem cells. ES cells have the ability to self renewal, proliferateimmortally and pluripotently differentiate into all kinds of tissues and organs. The discoveryof ES cells brings profound influence on researches on biological development regulationand biological tissue engineering. However, the studies on ES cells were strictly limitedbecause of the ethical and moral restraint.Embryonic carcinoma (EC) is a highly malignant tumor and occurs primarily in youngadults. EC cells, which are malignant stem cells of teratoma, have morphological andbiochemical properties similar to embryonic stem (ES) cells. Because of the similaritybetween ES cells and EC cells, ES researchers pay more and more attention on EC cells. InES cells, the regulation mechanism is very complicated and remains unclear. Furtherinvestigation on its regulation mechanisms is very critical for understanding thedevelopmental characters and practical significance correctly in the future.Wnt signal pathway plays great role in ES cells, regulating the ES cell self renewal anddifferentiation. It regulates the expression s of stem-like genes such as Oct-3/4and Nanog,and the fate determination of ES cells in skin, nerve system and hematologic system. Inaddition, Wnt up-regulates the expression of BMP family proteins, and inhibits the ES celldifferentiating into the nerve system. TCF/LEF(T cell factor/lymphoid enhancer factor)family proteins, the terminal factors of Wnt pathway, possess a highly conserved HMGdomain and an amino-terminal β-catenin interaction domain. When Wnt-stabilized β-cateninwas accumulated, TCF proteins promoted transcription of downstream targets such as c-Mycand Cyclin D1. In the absence of stabilized β-catenin, TCF proteins functioned astranscriptional repressors by interacting with corepressor proteins, such as CtBP andGroucho.TCF family proteins consist of four members, TCF1, TCF2, TCF3and TCF4. Amongthem, the expression of TCF3is most abundant in ES cells. It has been showed that T-cellfactor-3(TCF3) formed a repressor complex with TLE2(Groucho2) and co-occupies thepromoters throughout the genome with Oct4and Nanog, which is an integral part of the coreregulatory circuitry in the ES cells with pluripotency. Loss of TCF3or activation of Wntpathway increased the expression of Oct4, Nanog and other ES transcription factors, andprovoked stem cells resistant to differentiation. In addition, ablation of TCF3in ES cellsmaintained cell self renewal in the absence of exogenous leukemia inhibitory factor (LIF)and delayed response to differentiation stimuli. These findings indicate TCF3is very improtant for ES cell proliferation and self renewal. However, the role of TCF3in EC cellsand reprogramming has not been investigated and the mechanism still remains unknown.The purpose of this study is to investigate the effects of TCF3on EC development andprogress and explore the possible mechanisms. It is the first time to observe the effects ofTCF3overexpression on EC cells, which is very important for better understanding theregulation mechanisms of ES cells and EC tumors.MethodsThe recombinant retrovirus vectors were constructed using molecular cloning technique,and F9cell line over-expressed TCF3was established using retrovirus package and infectionmethod. The overexpression of TCF3in stable cell line was confirmed by Western blot andRT-PCR analysis. Effects of over-expressing TCF3on F9cells were detected by cellcounting, MTT assay, soft agar colony formation assay and migration assay in vitro. Thetransplant tumor model was established subcutaneously using stable transfected cell lines.Effects of over-expressing TCF3on NPC tumors were observed by tumor growth curve,tumor weight, survival percentage and HE staining in vivo. Effects of TCF3overexpressionand TCF3silencing after RNAi on the protein expressions of Oct4were detected byRT-PCR and Western blot analysis.ResultsThe overexpression of TCF3significantly inhibited proliferation, colony-forming andmigration in F9EC cells by approximately30,45and30%, respectively in vitro. The in vivomouse model showed that the overexpression of TCF3significantly reduced tumor volume(36.4%) and tumor weight (34.8%), malignancy progression and local infiltration andprolonged the life span of tumor-bearing mice. Overexpression of TCF3significantlydown-regulated Oct4expression in F9EC cells. On the contrary, TCF3silencing after RNAiremarkably up-regulated Oct4expression in F9EC cells.ConclusionsOverexpression of TCF3significantly inhibits F9EC cell growth, which indicates thatTCF3is an inhibitor of the malignant phenotypes of embryonal carcinoma through theregulation of Oct4expression TCF3.