Study The Effect Of ZD55-IL24 On Tongue Squamous Cell Carcinoma

Background and objectiveDespite advances in surgery, radiotherapy, the survival rates of patients with tongue cancer has not significantly improved over the past several decades. Gene therapy for tongue cancer is currently under investigation based on oncobiology .There are several general strategies utilized in gene therapy approach to cancer, such as antisense RNA, suicide gene therapy, immuno-therapy. Gene delivery systems, including viral vectors and non-viral vectors, are vital to gene therapy. Viral vectors are utilized widely for its high transduction efficiency including retrovirus vectors, adenovirus vectors, adeno-associated virus vectors. Because of not integrating into the host genome, easily produced, the use of adenovirus vectors for the delivery of therapeutic genes is becoming increasingly prevalent in the field of gene therapy. To find new vectors is urgent because of the short expressing time of exogenous genes, delivered by the first and second generations of adenovirus vectors .And oncolytic adenovirus is an attractive one.Oncolytic adenovirus is the use of genetically engineered adenovirus that specifically kill tumor cells while sparing normals via its cytolytic replication cycle;Research capitalizes on the oncolytic adenovirus to infect tumor cells efficiently, replicate, kill the host cell to release the progency virions and to spread. Pre-clinical and clinical studies have clearly shown that oncolytic adenovirus is feasible, but not ideal. To enhance its anti-cancer effects, current research in this area is focused on re-engineering adenovirus to make oncolytic adenovirus carry therapeutic genes.By constructing a oncolytic adenovirus vector------ZD55, carrying IL24,weobserve the transduction effects and study its effects and mechanism on human tongue squamous cell carcinoma cell line Tca8113.Methods1. We transduced human tongue squamous cell carcinoma cell line Tca8113 with ZD55 — GFP ,which carrying green fluorescent protein, and observe the transduction effects under the fluorescence microscope and phase contrast microscope.2. Flow cytometric analysis of positive-GFP expressing.3. Detecting CAR expressing of human tongue squamous cell carcinoma cell line Tca8113 by Western blot.4. Measuring the effects of ZD55 — IL24 on human tongue squamous cell carcinoma cell line Tca8113 by MTT.5. Detecting the cytopathic effect (CPE)of ZD55 —IL24 on human tongue squamous cell carcinoma cell line Tca8113 by stained with crystal violet.6. After human tongue squamous cell carcinoma cell line Tca8113 infected with ZD55—IL24,we detected the expressing of caspase-3 with Western blot.ResultsThe results of fluorescence microscope showed that 48hours after human tongue squamous cell carcinoma cell line Tca8113 infected with ZD55—GFP, different fluorescence intensity could be seen at multiplicities of infection (m.o.i.) of 1,10,100.Corresponding with increasing in multiplicities of infection, fluorescence intensity increased. The results of phase contrast microscope showed that cells morphous altered obviously after infection. Flow cytometric analysis showed that corresponding with increasing in multiplicities of infection, the rate of fluorescence expressing increased.Measured CAR expressing of human tongue squamous cell carcinoma cell line Tca8113 with western blot. Compared with cell lines in several kinds of hematologic disease,*K562^ Kassumi, human tongue squamous cell carcinoma cell line Tca8113 showed much more CAR expressing.Measure the effects of ZD55—IL24 on proliferation of Tca8113 cells with MTT colorimetric method. The results showed that 5 days after Tca8113 infected with ZD55-IL24 ,at multiplicities of infection (m.o.i.) of 1, 10,100, ZD55-IL24 had a substantial growth inhibitory effect on Tca8113 cells and displayed a dose—dependent manner. And 3,5,7days after Tca8113 infected with ZD55—IL24, at multiplicities of infectionlOO, ZD55—IL24 had a substantial growth inhibitory effect on Tca8113 cells and displayed a time—dependent manner.Observed the CPE of ZD55-IL24, ZD55-GFP, Ad-IL24onTca8113 cells and NHLF by stained with crystal violet. The result showed that 5 days after infection ,compared with the controls, ZD55—IL24> ZD55—GFP had a substantial lethal effects on Tca8113 ,while Ad—IL24 had little effects on it. And cells which were infected with ZD55—IL24 survived less than that infected with ZD55—GFP at the same multiplicities of infection.5days after NHLF infected with ZD55—IL24, ZD55-GFP, Ad—IL24,compared with the control, CPE was not detectable.Detected the change of caspase-3 protein activity by western blot technology, after Tca8113 cells were infected with 100 MOIZD55—IL24 respectively (0> 12, 24 > 48h),obvious splice in caspase-3 could be detected. With the time taken in infection increased, the more obvious splice could be seen.Conclusions1. Oncolytic adenovirus ZD55 can transduce human tongue squamous cell carcinoma cell line Tca8113 successfully, and has high transduction efficiency.2. Oncolytic adenovirus ZD55—IL24 has a substantial growth inhibitory effect on Tca8113 cells and displays a dose—and time—dependent manner.3. Oncolytic adenovirus ZD55 has no CPE on NHLF.4. Oncolytic adenovirus ZD5 5 — IL24 can induce apoptosis of Tca8113.5. Oncolytic adenovirus ZD55 can specifically in Tca8113 probably because of Tca8113's p53 deficient-function.6. The ability of oncolytic adenovirus ZD55 to replicate in tumor cells specifically and express therapeutic genes might facilitate the gene therapy and make huge progress.