Abstract:

Ma Xinxin, Zhang Mengli, Li Lingjun, Cao Yuping, Wu Qiuju, Ma Pengcheng. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding author: Ma Pengcheng, Email: mpc815@163.com 【Abstract】 Objective To investigate the effect of adenovirus-mediated IL-24 （Ad-IL-24） gene expression on the apoptosis in a human squamous cell carcinoma cell line COLO 16, and to explore the underlying molecular mechanism. Methods Cultured COLO 16 cells were divided into two groups to be transfected with an adenovirus vector carrying the IL-24 gene （Ad-IL-24 group） or green fluorescent protein （Ad-GFP group）, while those receiving no treatment served as the control group. After culture for different durations, qPCR was performed to quantify IL-24 gene expression, methyl thiazolyl tetrazolium （MTT） assay to evaluate the proliferative activity of COLO 16 cells, flow cytometry to detect the apoptosis of COLO 16 cells, laser scanning confocal microscopy （LSCM） to observe the morphological changes of COLO 16 cells, Western blot to determine the levels of Bax and Bcl-2 proteins and to evaluate the activation of caspase-3, qPCR to determine the levels of Bax and Bcl-2 mRNAs, an immunofluorescence assay to observe the expression of Bax and Bcl-2 proteins. Statistical analysis was carried out by a two-sample t-test with the SPSS 19.0 software. Results MTT assay showed that the proliferation of COLO 16 cells in the Ad-IL-24 group was significantly inhibited as early as 4 days after the transfection; thereafter, the inhibitory effect increased in a time-dependent manner, and peaked on day 6 （P < 0.05）. However, there was no significant difference in cellular proliferative activity between the Ad-GFP group and control group （P > 0.05）. Flow cytometry revealed that the apoptosis rate was significantly higher in the Ad-IL-24 group （13.10% ± 0.92%） than in the control group （3.69% ± 0.36%, P < 0.05） and Ad-GFP group （3.39% ± 1.06%, P < 0.05）, but no significant difference was observed between the control group and Ad-GFP group （P > 0.05）. LSCM demonstrated that the apoptosis of COLO 16 cells was accelerated in the Ad-IL-24 group. The immunofluorescence assay, Western blot and qPCR all showed that the mRNA and protein expressions of Bax were increased, but those of Bcl-2 were decreased in the Ad-IL-24 group compared with the Ad-GFP group and control group. Moreover, Western blot showed a protein band that could specifically bind to the anti-cleaved caspase-3 antibody in the Ad-IL-24 group, but not in the Ad-GFP group or control group. Conclusions Ad-IL-24 can induce apoptosis in human COLO 16 squamous cell carcinoma cells, probably by up-regulating Bax expression, down-regulating Bcl-2 expression, and activating caspase 3.