Inhibition Effect Of Exogenous IL-24 Gene On Rat C6 Glioma Cells

Objective: Malignant brain tumor has become one of the main diseases which lead to death among the malignant tumor. It has become the second death inducing factor in the group under 15 among people died from cancer. The results are fourth for men in 35-54 ages'group and women in 15-34 ages'group. Malignant gliomas are the most common brain tumors in humans. Current treatments failed to provide long-term management of these tumors. The prognosis for patients with high-grade glioma remains poor: survival for less than 1 year, even following surgery and adjuvant therapies such as chemotherapy and radiation therapy. It is very clear that malignant glioma is a severer disease which is prone to occur during productive age and how to improve the therapeutic efficacy is a challenging subject facing to each surgeon.This disease clearly requires development of more effective therapeutic strategies that will improve long-term control and survival. The development of gene therapy techniques has provided a new and promising avenue of research into more efficacious treatments for gliomas and other cancers.IL-24/MDA-7(The melanoma differentiation associated gene-7, mda-7) is a new gene originally cloned by Jiang H. from differentiated human malignant melanoma HO-1 cells induced by IFN-βand MEZ which belongs to PKC activating agent.IL-24/MDA-7 is a dual-function molecule with tumor suppression and cytokine functions. Studies have proved that IL-24/MDA-7 has wide tumor suppression function in diversity malignant cancers including proliferation inhibition, apoptosis induction, neovascularization, radiosensitization effect. Recent studies also found invasion and metastasis of malignant tumor are inhibited by IL-24/MDA-7 by lowering the expression of some related genes.We aimed to observe the inhibition effect of IL-24/MDA-7 on C6 glioma cells by studying C6, C6/IL-24, and C6/pLXSN cells. The expression of cyclin B1, survivin, p53, Membrane Typy MMP 1 (MT1-MMP, MMP-14) mRNA and proteins are tested to investigate the mechanisms of the anti-tumor effect of IL-24/MDA-7.Methods:1. Cell cultureThe C6 cells, C6/IL-24 cells and C6/pLXSN cells were cultured with the RPMI1640 culture medium containing 10% fetal calf serum (FCS), penicillin 100 U/ml, streptomycin 100μg/ml at 37℃in humidified 5% CO2 incubator.2. Cell viability measured by MTT assay in vitroThe log-phase C6/IL-24, C6/pLXSN and the C6 cells were detachated with 0.04% ethylenediamine tetraacetic acid (EDTA) and seeded in 96-well tissue culture plates respectively at 200μl/well of 1.5×104/ml cell concentration, incubated at 37℃in humidified 5% CO2 incubator for 6 days. Absorbance was measured at 490 nm wavelength every day. 20μl/well of 5 mg/ml MTT solution was added and incubated for 4 hours. After centrifugalization the MTT (Methyl thiazolyl tetrazolium) solution and cell supernatant was removed and the cells and dye cristals were dissolved by adding 150μl/well of dimethylsulf- oxide (DMSO). Absorbance was measured at 490 nm wavelength with ELX800 microplate reader. The assays were repeated three times. The results were expressed as mean±s and growth curves were drawn.3. The distribution of cell cycle and apoptosis rate detected by flow cytometry (FCM)The cells were collected, fixed with 70% ethanol for 24 hours at 4℃, and stained using EB for 30 minutes. The cell cycle was detected using flow cytometer.4. Transwell chamber invasion assay in vitroInvasion assays were performed in Transwell chambers supplied by Corning Corporation. According to the supplied instruction, the lower chamber were filled with 500μl cell cultural medium containing 10% FBS. 300μl of C6/IL-24 or C6/pLXSN or C6 cell suspension (1×105/ml) were added into each of the upper chamber after rehydration of Matrigel Matrix. The polycarbonate membranes with 8μm pore coated with Matrigel Matrix were placed between upper chamber and lower chamber. The Transwell chambers were then incubated at 37℃in 5% CO2 for 36 hours. After incubation, taking off the membrane, cells on the upper side of the membrane were removed by scraping. The membranes were placed on slides, fixed with methanol, and then stained with haematoxylin, dehydrated with alcohol gradually, after transparence with xylol sealed with neutral gum. Cells on the lower side of the membrane were counted in four random fields of high magnification for each membrane.5. Expression of cyclin B1, survivin, p53 and MT1-MMP mRNA detected by RT-PCRPrimers of cyclin B1, survivin, p53 and MT1-MMP andβ-actin were designed with Premier 5.0 software according to their nucleotides sequence. Total RNA was extracted from C6/IL-24, C6/pLXSN and C6 cells with Trizol reagent. 2 microgram of RNA was reversely transcribed using random hexamer primers in a thermocycler (37℃for 60 minutes and 95℃for 5 minutes). After predenaturation at 95℃for 10 minutes, cDNA amplification was performed. PCR amplification products were separated in a 1.50% agarose gel, photographed and analysed with gel image analysis system. Data are presented as the ratio of the integrated optical density (IOD) of cyclin B1, survivin, p53 and MT1-MMP (MMP-14) to that ofβ-actin acting as an internal standard.6. Expression of cyclin B1, Survivin, p53 and MT1-MMP protein detecked by western blotting assay The protein was abstracted on ice from C6/IL-24, C6/pLXSN and C6 cells lysed in buffer. Protein concentration was determined using Coomassie brilliant blue G250 protein assay kit. Samples (50μg) were separated by SDS-PAGE and were transferred to a nitrocellulose filter (NC filter). The membrane was blocked for 1 hour at room temperature with blocking solution containing 5% nonfat milk and then incubated with primary antibody overnight at 4℃. The membrane was washed and incubated with secondary antibody (goat antimouse IgG-horseradish peroxidase) at 37℃for 1 hour and then washed again and developed using the ECL (enhanced chemilumine- scence) kit according to the supplier's instructions. The membrane was photographed and then analysed with gel image analysis system expressed as the ratio of the integrated optical density (IOD) of cyclin B1, Survivin, p53 and MT1-MMP (MMP-14) to that ofβ-actin acting as an internal standard.7 Statistical analysesThe results were expressed as mean±standard deviation and evaluated with SPSS 13.0 solftware by analysis of variance (One-Way ANOVA). P <0.05 was consided statistically significant.Results:1. Cell viability measured by MTT assayThe C6/IL-24 cells can proliferate in vitro. But compared with the C6/pLXSN and parental C6 cells, the absorbance of C6/IL-24 cells was decreased; suggesting the proliferative ability of C6/IL-24 cells was attenuated.2. The distribution of cell cycle and apoptosis rate by FCMThe G0/G1 phase of C6/IL-24 cell, C6/pLXSN and C6 cell are 58.42±3.12%, 49.04±3.14% and 51.41±4.69%; S phase cells are 14.51±1.23%, 43.63±3.09% and 42.25±2.45%; the G2/M phase cells are 27.07±2.17%, 7.33±1.03 and 6.34±1.05%. The G2/M phase cells of C6/IL-24 were increased as compared with C6/pLXSN and C6 cells(P<0.05), but C6/pLXSN versus C6 cells was no significant difference(P>0.05). This result suggests that G2/M cell cycle arrest exists in C6/IL-24 cells. There was significant apoptosis peak in the cell C6/IL-24, which owned an apoptosis rate of 15.46±1.78%. There was no apoptosis peak in C6/pLXSN or C6 cell, which owned 4.37±0.51% and 4.04±0.52% apoptosis rate respectively. These results showed higher apoptosis rate in C6/IL-24 cell than C6 or C6/pLXSN cell (P<0.05).3. Invasion assay in vitro by TranswellThe number through polycarbonate membrane in average visual field was 8.31±0.88, 14.65±1.08 and 15.08±1.18 in C6/ IL-24 cells, C6/pLXSN cells and C6 cells respectively. There was statistical significant difference between the number of C6/ IL-24 cells through the membrane and the number of C6/pLXSN cell or C6 cells (P<0.05). There was no statistical significant difference between the number of C6/pLXSN cells and C6 cells(P>0.05). 4. Expression of cyclin B1, Survivin, p53 and MT1-MMP (MMP-14) mRNAThe ratios of IOD between cyclin B1 andβ-actin mRNA were 0.58±0.04, 1.19±0.04 and 1.14±0.05 in C6/IL-24, C6/pLXSN and the parental C6 cells respectively, those between survivin andβ-actin were 0.91±0.05, 1.39±0.05 and 1.44±0.04, those between p53 andβ-actin were 1.29±0.05, 1.34±0.04 and 1.36±0.07; those between MT1-MMP andβ-actin were 0.98±0.04, 1.52±0.09 and 1.64±0.09.The cyclin B1, survivin, MT1-MMP mRNA expression of C6/IL-24 cells was down-regulated (P<0.05), but there was no significance between C6/pLXSN and C6 cells(P>0.05). There was no significance of the mRNA expression of p53 between C6/IL-24, C6/pLXSN and C6 cells(P>0.05).5. Expression of cyclin B1, Survivin, p53 and MT1-MMP protein detecked by western blotting analysisThe ratios of IOD between cyclin B1 andβ-actin protein were 0.49±0.04, 0.65±0.05 and 0.70±0.03 in C6/IL-24, C6/pLXSN and the parental C6 cells respectively, those between survivin andβ-actin were 0.14±0.02, 0.40±0.04 and 0.42±0.05, those between p53 andβ-actin were 0.55±0.04, 0.58±0.06 and 0.54±0.04; those between MT1-MMP andβ-actin were 0.20±0.01, 0.40±0.03 and 0.43±0.04. The cyclin B1, survivin, MT1-MMP protein expression in C6/IL-24 cells was down-regulated (P<0.05), but there was no significance between in C6/pLXSN and C6 cells(P>0.05). There was no significance of the protein expression of p53 in C6/IL-24, C6/pLXSN and C6 cells(P>0.05).Conclusions:1 The exogenous IL-24 gene can affect the biological characteris- tic of the C6/ IL-24 cells and suppresse the proliferation of the C6/ IL-24 cells.2 The exogenous IL-24 gene expressions could induce G2/M cell cycle arrest which may be closely related to the down regulated expression of cyclin B1 in C6/IL-24 cells.3 Proliferation inhibition and apoptosis induction effects of IL-24 on C6/IL-24 cells are related to down-regulation of survivin,whose expression is independent of the expression of p53.4 IL-24 may repress invasion of C6 glioma cells through down-regulation of MT1-MMP.