Experimental Study Of The Effect Of FHIT Gene Transfection In SW480 Human Colon Carcinoma Cell Growth

Colon Carcinoma becomes more and more commonly seen and the death caused by it is increasing worldwide recently. Surgery is the most important treatment method but chemotherapy failed to significantly benefit the patient after the surgery. Therefore, in order to increase survival rate and improve life quality, searching for method of early diagnosis and treatment is of most importance. Recent development in tumor immunology and molecular biology has shed some light on the mechanism of the occurrence and transferring of the Colon Carcinoma, which provides basis to gene therapy. Preliminary experiments have shown that the failed expression of Fragile Histidine Triad (FHIT) play an important role in the initiation and development of the Colon Carcinoma, but its application in gene therapy is still under satisfaction; many of them are still under clinical experiment phase. Moreover, due to the complication of the expression of the human genes, in-depth research is needed to elucidate some unknown factors, in order to fully utilize the advantage of the gene therapy. Once the mechanism of the cancer cell death due to transfection of FHIT into Colon Carcinoma cells is elucidated, new strategies of gene therapy of Colon Carcinoma can be formulated.This research aim to employ cationic liposome Lipofectamine 2000 to trasfect eukaryotic vector pFHIT, obtained through gene engineering, into Human Colon Carcinoma cell series SW480. Via G418 screening, paper disc method combined with optimal concentration, we can select the stable mono cloned transfected gene, test for SW480-FHIT, explore the degree of expression of FHIT in SW480-NEO cells after transfection of pRc/CMV2, as well as in wild type SW480 cells. We also observe the effect of FHIT on cell growth in vitro, establish the SW480 Colon Carcinoma pathological model in BALB/C NU naked mouse, and discuss the antitumor effect of FHIT.PartⅠUtilization of Gene Transfection to Establish Stable SW480 Transfectant of FHITObjectiveUtilization of Gene Transfection to Establish Stable SW480 Transfectant of FHITMethod1. Cell culture SW480 Colon Carcinoma cells were maintained in complete high glucose DMEM medium containing 10% fetal bovine serum and 2mM L-Glutamine.2. Gene transfer into cells, G418 selection and Clone of the stable transfectants SW480 Colon Carcinoma cells were transfected with pFHIT (constructed by former group member Ping Yang) and pRc/CMV2 via cationic liposome Lipofectamine 2000. After 48 hours, a series of complete DMEM dedium with gradient elevated concentration of G418 was used to culture and select the transfected cells for three to four weeks, including a two week selection period with optimal concentration of G418. G418-resistant Colon Carcinoma cells were then cloned via optimal concentration method.3. PT-PCR and Western Blot for detecting FHIT expression Total RNA was extracted from all the stable transfectants and their parental wild type Colon Carcinoma cells using TRIzol reagent. UV absorbance and 1% formaldehyde denatured gel electrophoresis were done. And expression of the protein was detected by Western Blotting.Result1. Identification of the recombinant plasmid Use FHIT primer to detect FHIT fragment. PCR products were run on a 1% agarose gel stained with Goldview and visualized by UV illumination. Target band showed up at around 431bp in the FHIT lane, while the parental pRc/CMV2 had no such band.2. Gene transfer into Colon Carcinoma cells, G418 selection and clone of the stable transfectantsTransfection through Lipofectamine 2000 can achieve satisfied outcome with a mild disturbance to cells. Complete DMEM containing 800μg/ml G418 is optimal for screening transfected Colon Carcinoma cells. Through G418 selection and limiting dilution of resistant cells, stable transfectants were cloned.3. FHIT expression evaluationUsing FHIT reconstructed plasmid, pRc/CMV2 and cDNA in blank SW480 as primer to run RT-PCR. The sample transfected with FHIT reconstructed plasmid shows FHIT band, whereas the blank and control experiments do not. Western blot has detected FHIT expression in FHIT transfected cells, whereas the blank and controls cells showed negative results.ConclusionTransfection through Lipofectamine 2000 can achieve satisfactory outcome with a mild disturbance to cells. Through G418 selection and limiting dilution of resistant cells, stable FHIT transfectants can be cloned. These transfectants can express FHIT whereas the vector control transfectants and the parental cell line have no FHIT expression. Part II In Vitro Proliferation Study of FHIT Expressing Transfectant MethodsObjectiveDetermination of effects of FHIT towards growth of SW480 Human Colon Carcinoma cells in votro by MTT and flow cytometer.Method1. Grouping For each stable transfectant category, one cell line was selected and named as experimental SW480-FHIT (FHIT transfected) and vector control SW480-NEO (pRc/CMV2 transfected). Parental SW480 cells were used as blank control.2. MicroscopyThree groups of cells were observed for their morphological changes under light microscopy.3. In vitro proliferation assay and cell growth chart 1×104 cells were plated into each well on the 24-well plates in triplicate and cultured for 7 days. Every 24h after seeding 3 wells of cells were collected and counted, the average number of live cells was recorded. The cell growth curve was then charted. Differences among average cell number of each group each day were compared through one-way ANOVA analysis using a SPSS13.0 software package.4. Flow cytometry (FCM) was used to detect the cell apoptosis and cycle.The cell were plated into 6-well plates, and collected when confluensing to 90%. After modulating the cell density to 1×106/ml and coloured by PI, use flow cytometry to detect the cell apoptosis and cycle.Result1. Cell growth by microscopyUnder normal culture condition, three groups of cells have no significant differences in their morphology and growth pattern, as determined by microscopy.2. Cell growth before and after FHIT transfection by MTTFHIT transfected cells showed noticeable decrease in growth rate, compared to SW480-NEO and parental SW480. This decreasing in growth rate is even more significant when the cell growth reaches exponential increasing phase (P<0.05).3. Cell cycle and death ratio of SW480 before and after FHIT transfection SW480-FHIT has significantly lower ratio of G2/M phase and S phase; more cells were in G0/G1 phase. Moreover, the death rate is higher than the rest of the two groups (P<0.05)Conclusion FHIT can significantly reduce the growth of the Human Colon Carcinoma cells, suppress their growth cycle and increase their apoptosis rate.PART III In Vivo Study of the Antitumor Function of FHIT MethodsObjectiveDetermination of effects of FHIT towards growth of SW480 Human Colon Carcinoma cells in naked mouse. Preliminary experiments towards elucidating the suppression mechanism.Method1. Grouping of animals 60 BALB/C NU mice (male, 4 to 5 weeks) were purchased and equally randomized into 3 groups. Each group received SW480-FHIT, SW480-NEO and SW480 inoculation, respectively, as it was named.2. Establishing animal model of tumor-bearing Three cell lines were cultured to the log phase with sufficient cell number, then harvested and resuspended to 5×107cells/ml in PBS. Each mouse was inoculated with 0.2ml of cell suspension (1×107 cells) subcutaneous in the right flank.3. Observation of established tumorFormation and growth of the tumor was observed. The longest (a) and shortest (b) diameters of the established tumor were measured with a caliper from the 10th day after inoculation and repeated every 2 days. The tumor volume was calculated as 0.5×a(mm)×b2(mm). Ten randomly selected mice were executed and tested when tumor volume had reached 500 mm3; the rest were kept alive and used for recording the survival time, in order to compare the life time of the tumor bearing mice. Statistical analysis was carried out with a SPSS13.0 software package.4. Detection of tumor sample.The tumors of BALB/C NU mice were sampled. Expression of FHIT mRNA was detected by RT-PCR, and expression of the protein was detected by Western Blotting.Results1. The effect of FHIT on tumor transplanted in BALB/C NU mice Three groups of mice all formed palpable subcutaneous tumor in 7~10 days after inoculation. SW480-FHIT tumor grew slower than the tumors from those two control groups according to the tumor growth curve. Differences were statistically significant compared by one-way ANOVA analysis (SW480-FHIT vs. SW480: p<0.01; SW480-FHIT vs. SW380-NEO: p<0.01), whereas tumors from the two control groups grew at about the same rate (p>0.05).2. The effect of FHIT on life time of the tumor bearing miceThe life time of SW480 group was 35 days, 95% CI (30.868,39.132); the life time of SW480-NEO group was 37 days, 95% CI (35.482, 38.518); the life time of SW480-FHIT group was 48 days, 95% CI (43.868, 52.132). The average life times for the afore-mentioned three groups were 34.600±1.558, 36.100±1.479 and 50.700±3.685 days, respectively. The difference between the experimental group and the control group were statistically meaningful (p<0.01). 3. FHIT expression in tumorRT-PCR results showed that the tumor cells in SW480-FHIT group have varies amount of FHIT expression. Western Blot had confirmed that the FHIT protein was expressed. In contrast, mRNA and protein level test were FHIT negative in the controls groups. P53 analysis showed that SW480-FHIT group has the highest P53 expression compared to the control groupsConclusion1. FHIT can significantly suppress the growth of the tumor cells in vivo and elongate the survival time of the tumor-bearing mice.2. FHIT transfected SW480 cells showed FHIT expression, and P53 expression was significantly increased, which implies that FHIT suppress growth of tumor cells by increasing the expression level of P53.