He Jin-hua, Zhang Xiao-ying, Wu Feng-yun, Liao Xiao-li, Wang Wei, Jiang Jian-wei
Department of Biochemistry, Medical College, Jinan University, Guangzhou 510630, China.
Yao Xue Xue Bao. 2011 Feb;46(2):138-45.
In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P < 0.05). After Lovo cells treated with apollon ASODN for 48 hours, the expression of the apollon mRNA level was suppressed significantly. And a marked concentration-dependent decline of cell proliferation and clone forming, increasing of cell apoptosis levels were observed. The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry. Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation. Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon can suppress the expression of apollon mRNA, and inhibit the proliferation, induce apoptosis, arrest cell cycle at S phase of colorectal cancer Lovo cells in vitro and enhance the chemo-sensitivity to 5-FU, DDP and EPI.
本研究探讨了阿波隆反义寡脱氧核苷酸（ASODN）对人Lovo细胞体外增殖和凋亡的影响。将阿波隆ASODN与人结肠直肠癌Lovo细胞孵育48小时，分别采用WST法和克隆形成试验检测增殖抑制率和克隆形成率。通过实时荧光定量逆转录聚合酶链反应分析阿波隆mRNA的表达。采用流式细胞术测定凋亡细胞百分比和细胞周期分布。用荧光显微镜观察凋亡细胞的形态。将阿波隆ASODN与不同浓度的5-氟尿嘧啶（5-FU）、顺铂（DDP）或表柔比星（EPI）联合孵育Lovo细胞，用WST法检测细胞增殖抑制率并计算IC50。结果发现，靶向阿波隆基因的ASODN均能显著抑制Lovo细胞的生长并诱导其凋亡（P<0.05）。阿波隆ASODN处理Lovo细胞48小时后，阿波隆mRNA水平的表达明显受到抑制。观察到细胞增殖和克隆形成呈明显的浓度依赖性下降，细胞凋亡水平增加。流式细胞术检测显示，G0/G1期细胞百分比降低，S期细胞百分比增加，Lovo细胞停滞于细胞周期S期。许多用Hoechst 33258染色的Lovo细胞呈现出凋亡形态，如细胞收缩、核浓缩和核碎裂。检测到细胞增殖抑制，与单独的5-FU、DDP和EPI组相比，阿波隆ASODN（0.08 μmol·L-1）分别增强了5-FU、DDP和EPI对Lovo细胞的化疗效果，敏感性分别提高了约2.58、4.47和5.
