Therapeutic Effect Of Interferon-α Gene By Recombinant Adeno-associated Virus In Xenograft Metastasis Models From Nasopharygeal Carcinoma Cell

Nasopharyngeal carcinoma (NPC), a type of head and neck cancer, shows adistinct racial distribution among Chinese living in South China and and SoutheastAsia.The 5-10 years survival rate of advanced NPC patients is only 40%. The majorfactors affecting the survival of NPC patients include local recurrence andmetastases. Besides this, a serious side-effect was observed in patients with currentionizing-radiation therapy and chemotherapy, which affect the living quality ofNPC patients. It is critical to develop some novel therapies, which can enhanceoverall survival rate and improve living quality by preventing and antagonizingmetastases in NPC patients.Biotherapy has been becoming progressively the fourth model in controlingmaligmant tumor with development of tumor biology. Biotherapy of tumor playsthe key role in general treatment, especialy in targeting recurrence and metastases.Interferon(IFN), one of major biotherapic medicine, has been used worldwidely forthe treatment of more than 14 types of cancer, including some hematogenousmalignancies and some solid tumors.IFN has diverse biological functions, includingsurppressing tumor related virus, inducing differentiation of tumor cells, promotingapoptosis of tumor cells, inhibiting proliferating of tumor cells, immunomodulatingimmune systems of patients and inhibiting angiogenesis of tumor. However, there is a poor understanding about effects of IFN on NPC. In the present study, we willinvestigate the effects of IFN on proliferative activity and metastases formation inEBV positive NPC tumor cells in vitro and in vivo.Pharmacokinetics studies indicated that IFNs exhibit an extremely shorthalf-life in the blood system after parenteral adminstration. Injections of high dosesof cytokines repeately, apparently needed to achieve effective antitumor response,often cause the adverse effects on the human body. In some clinical trials, somepoor response with cancer tissue were observed on patients due to an insuficientdelivery of the cytokine to the correct target site.Thus, some alternative strategies ofstable, effective and targeted delivery systems of INFs are urgently needed to bedeveloped. In this study, we constructed a recombinant adeno-associated viralvector harboring interferon alpha gene (rAAV-IFN-α),demonstrating efficient andcontinous expression of active IFN-αprotein.The antitumor effects of IFN-αgenedelivered by this new strategy are much better compared to that by conventionalclinical use of IFN-αprotein.Our results may suggest a novel therapeutic approachfor treatment of patients with advanced NPCConstruction of the recombinant adeno-associated virus(rAAV) and AssayPlasmid pAAV-IFN-αwas constructed by inserting IFN-αgene into BamH I andEcoRI sites of plasmid pAAV using Bglâ…¡â… /EcoRI enzymes.Plasmid pAAV-EGFPwas similarly constructed by inserting EGFP gene between the XhoI and EcoRI siteof pAAV. Recombinant AVV particles were produced by using a helper virus-freesystem.rAAV vectors and helper plasmid pDG were co-transfected into HEK 293-FTcells by calcium phosphate precipitation method.The supernatant fraction containingrAAV-IFN-αor rAAV-EGFP particles was decanted.rAAV particles were purified byHiTrap Heparin column chromatography.The final titer of the purified viral vectors was quantified by real-time PCR.The concentration of viral particles ranged from 10¹¹ to 10¹² v.g./ml. After C666-1cells, a kind of EBV-positive NPC cell line, were infected with rAAV-EGFP atdifferent concentration at 48 hr, the highest transfective efficiency (95%) was obtained with 5×10⁴ v.g./cell multiplicity of infection (MOI).Transfective efficiencyof the purified viral vectors was maintained.rAAV-IFN-αinfected C666-1 cellsshowed the stable expression of IFN-αby RT-PCR and Western blot assay.The resultssuggested the higher transfective efficiency of rAAV-IFN-α.Effect of rAAV-IFN-αon EBV-positive NPC cell lineA series of experiments were performed to study effects of IFN-αonEBV-positive NPC cell line C-666-1.These methods included half-percent cell growthsuppressed(IC₅₀) dose test of IFN-α, MTT assay for cell proliferation, flow Cytometryassay and apoptosis assay, detection of IFN-αand metastases-related genes inC-666-1 cell infected rAAV-IFN-αby RT-PCR and Western bloting, measurement ofIFN-αlevel in cell culture supematant by liquichip technique.The results showed that C-666-1 were relatively resistant to antiproliferativeeffects of IFN-α,whose IC₅₀ was 1×10⁵IU/ml. To determine the effect ofrAAV-IFN-α-infected C666-1 cells on growth,antiproliferative activity of withHighest transduction efficiency (95%) had not revealed for 48 hr, which opticaldensity(OD) was no statistically significant compared to control (F=1.181,p=0.334).Until 72hr rAAV-IFN-α-infected C666-1 cells growth had not beensuppressed. Flow Cytometry assay showed a significant increasing percentage ofrAAV-IFN-α-infected C666-1 cells in the G₁ phase compared to therAAV-EGFP-infected C666-1 cells in the G₁ phase (F=39.675,P=0.000).The effectof rAAV-IFN-αon cell cycle was better than that of IFN-αprotein bymulti-comparison, but no statistically significant. In vitro, no apoptosis wasobserved in rAAV-IFN-α-infected C666-1 cells or IFN-α- incubated C666-1 cells.By RT-PCR.assay, we found that the mRNA expression of LMP-1 and EBNA-1 inEBV-positive C666-1 cells were suppressed, and the mRNA expression MMP-9 andVEGF were down-regulated by rAAV-IFN-α.. IFN-αprotein of IC50 had the sameefffect on C666-1 cells but it was weak. The results of Liquichip test showed theamounts of IFN-αin culture supematant of rAAV-IFN-αinfected C666-1 cell wereelevated with increasing incubated cell time,but the total concentration of IFN-α measured at 72 hr was less than 20U/ml.Effects of rAAV-IFN-αand IFN-αon growth and metastases of establishedxenograft EBV-positive NPC tumor in vovo64 male nude mice (BALB/c nu/nu) were used to establish liver xenograftEBV-positive NPC matastases model by direct injection of 1×10⁶/ml C666-1 cellsinto the liver.The 64 nude mice were divided into 4 groups of 16,A group withrAAV-IFN-α, B group with IFN-α, C group with rAAV-EGFP and D group with PBs.8 mice from each group were randomly chosen to evaluate the therapeutic effects andwere sacrificed at the third weekend of C666-1 cell injection.The remainder were leftto evaluate the long-term survival time until die naturelly.The survival time of theremainder mice were recorded, and a survival curve was constructed and analyzed bya log-rank test.Effect of rAAV-IFN-αand IFN-αon growth and metastases ofxenograft NPC were observed in vovo.Immunohistochemical assay was used to detectthe expression of MMP9 and MVD.The apoptosis of xenograft NPC tissue wasanalyzed by TUNEL staining. RT-PCR was performed to test the mRNA level ofIFN-α,MMP9 and VEGF in transplanted tumor.Liquichip technique was applied tomeasure the amount of human IFN-αand murine IL-12,GM-CSF,IFN-r,IL-2,IL-10,TNF-αin the mice serum. The data were Statistically analyzed by SPSS10.0.The results showed the volume of the transplanted tumor in 4 groupsrespectively were (239.00±44.80)mm³,(340.25±75.96)mm³,(424.75±67.22)mm³,(430.75±112.05)mm³ after implanting directly C666-1 cells into the liver for 3weeks.The volumes of the transplanted tumors in 4 groups differed significantly(F=10.397,P=0.000).rAAV-IFN-αgroup had a significant differences with othergroups by mul-comparesion (P=0.000).Satistical difference were significant, whichwere found between IFN-αand the controls (P=0.029) but not between group C andgroup D. Metastases rate in liver were 12.5%,37.5%,100%,100% and metastasesrate in lung were 0.0%,50.0%,87.5%,100% in group A,B,C,D respectively. Theaverage survival time in 4 groups were respectively (18.25±2.49)days, (15.13±1.13))days,(12.88±1.25))days,(12.63±1.19))days. Survival analysisdemonstrated satistical significance among 4 groups (F=20.848,P＜0.001).Survivaltime of rAAV-IFN-αwas much longer than IFN-αgroup (P=0.001) and 2 controlsgroups (P=0.000). Furthermore, survival time of IFN-αgroup was longer than twocontrol groups (P＜0.01).The apoptotic indices for rAAV-IFN-α, IFN-α,rAAV-EGFP,PBS groups were calculated to be (12.20±1.92),(8.80±1.64),(4.00±1.58),(3.40±1.14), There were significant differences among 4groups(F=34.118, P=0.000).Group A had a significant differences compared to theother 3 groups by mul-comparison.Satistical difference were also found betweenIFN-αand the controls (P=0.000),but not between rAAV-EGFP and PBS groups.Immunohistochemical analyses on rAAV-IFN-αand IFN-αgroups showed thatthe MMP9 expression was down-regulated compared to rAAV-EGFP and PBS groupsin the transplanted tumor of liver tissue,which had satistical significance (P=0.000).Immunohistochemical detection of CD31 revealed significantly a lower microvesseldensity in rAAV-IFN-α(1.60±1.14) and IFN-αgroup (1.80±0.84) than that in theEGFP (13.00±2.74) or PBS (13.20±2.59) groups respectively; which had asignificant difference (F=53.498, P=0.000).Additionally, the expression of IFN-αmRNA was confirmed in the transplanted tumor of rAAV-IFN-αgroup, but not foundin the other groups.The expression of MMP9 mRNA and VEGF mRNA intransplanted NPC were decreased in group A and B compared to controls.We examined the amounts of IFN-αin the serum produced from intravenousrAAV-IFN-αbefore 3 weeks or from subcutaneous injection of IFN-αfor 3 weeksThe results showed that the serum IFN-αlevel(U/ml, (?)x±s) in rAAV-IFN-αgroup(7.01±2.68) was higher than IFN-αgroup (5.59±1.42),which had no satisticaldifference (P=0.126).There was a significant difference in the amounts of IFN-αofserum between treatment groups and controls (1.81±0.48),(1.30±0.34) (p=0.000).To confirm the cytokine induction by rAAV-IFN-αand IFN-αused in this study, wefound the level of IL-12,GM-CSF were increased after treatment.The serum IL-12 level(pg/ml,(?)±s) were respectively (105.02±43.43),(56.99±13.93),(0.00±0.00),(0.00,0.00),which had the satistical difference Between treatment groups andcontrol groups (P=0.002). The serum GM-CSF level (pg/ml,(?)±s) were respectively(105.23±21.05),(100.52±14.59),(72.42±22.76),(63.08±26.100), which satisticaldifference was not found between group A and group B but between treatment groupsand control groups.The levels of IFN-r,IL-2,IL-10,TNF-αin serurn were alsomeasured and found no significant differences between treatment groups andcontrols.Conclusion1,Our results suggested that the growth and metastases of liver transplantedNPC were suppressed efficiently and that survival time was prolongedremarkblely after treatment by intravenous rAAV-IFN-αor subcutaneousinjection of IFN-α, The effects on antiprolification,metastasticsuppression,survival time and apoptosis analysis with rAAV-IFN-αdeliverywas more powerful than that of IFN-αprotein delivery, rAAV-mediatedIFN-αgene therapy is a promising strategy in suppressing growth andmetastases of EBV-positive NPC tumor.2,The effects of IFN-αon EBV-positive NPC may be attributed toantiangiogenesis and immunomodulatary role in this experiment.But it islimited that antitumor direct response of IFN-αhad been observed.3,The effects of IFN-αon suppressing growth and metastases of EBV-positiveNPC tumor.may be obtained by inhibiting expression of LMP-1 andEBNA-1 in EB virus.