Fang Yongqi, Sun Hongli, Zhai Jing, Zhang Yuanying, Yi Shuying, Hao Gangping, Wang Tao
School of Basic Medicine, Taishan Medical University, China.
Asian Pac J Cancer Prev. 2011;12(10):2721-6.
Nuclear factor-kappaB (NF-kB), a transcription factor, is abundantly expressed in prostate cancer and regulates many tumor-related genes. Given the important roles of these genes in tumor control, the present study was conducted to test the hypothesis that there was different expression of NF-kB in androgen- dependent or androgen-independent prostate cancer cells. In addition NF-kB decoy oligodeoxynucleotides (ODNs) were transfected into two prostate cancer cells to determine affects on growth and apoptosis.
First, NF-kB decoy ODNs were designed according to the NF-κB elements in the promoter region of c-myc gene. Then, NF-kB and control decoy ODNs were transfected with lipofectamine. Their influence on prostate cancer cell line proliferative activity was detected by MTT assay. Cell apoptosis was determined by flow cytometric(FCM) analysis and AO/EB study. Thirdly, nuclear extracts were prepared from PC-3M cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). Lastly, to confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kB decoy ODNs into PC-3M cells with lipofectamineTM2000. Expression levels of related endogenous genes were assessed by western blotting.
We found overexpression of NF-kB in the androgen-independent prostate cancer cell line PC-3M compared to the androgen-independent LNCaP. Treatment with NF-kB decoy ODNs resulted in strong suppression of proliferation, especially in the PC-3M case. Induction of apoptosis of PC-3M was observed in FCM and AO/EB studies. Activity of luciferase was significantly reduced in the NF-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that expression of some endogenous genes was reduced, while other genes transcripts were induced. EMSA demonstrated specific binding of the NF-kB decoy to NF-kB protein.
These findings indicate that NF-kB activation plays an important role in evolution of androgen-independent prostate cancer via manipulating expression of target genes. Inhibitors of NF-kB may thus offer promise as a therapeutic approach for the treatment of androgen-independent prostate cancer. NF-kB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.
核因子-κB（NF-κB）作为一种转录因子，在前列腺癌中大量表达并调控许多肿瘤相关基因。鉴于这些基因在肿瘤控制中的重要作用，本研究旨在验证雄激素依赖性或雄激素非依赖性前列腺癌细胞中NF-κB表达存在差异这一假说。此外，将NF-κB诱饵寡脱氧核苷酸（ODNs）转染至两种前列腺癌细胞中，以确定其对细胞生长和凋亡的影响。
首先，根据c-myc基因启动子区域的NF-κB元件设计NF-κB诱饵ODNs。然后，用脂质体转染NF-κB诱饵ODNs和对照ODNs。通过MTT法检测它们对前列腺癌细胞系增殖活性的影响。采用流式细胞术（FCM）分析和AO/EB染色法测定细胞凋亡情况。第三，从PC-3M细胞中提取核提取物，通过电泳迁移率变动分析（EMSA）检测DNA-蛋白质相互作用。最后，为了确定作用机制，构建了一个含有c-myc启动子片段的pGL3-C-MYC荧光素酶表达载体，并与NF-κB诱饵ODNs用脂质体TM2000共转染至PC-3M细胞中。通过蛋白质印迹法评估相关内源性基因的表达水平。
我们发现，与雄激素依赖性的LNCaP细胞相比，雄激素非依赖性前列腺癌细胞系PC-3M中NF-κB过表达。用NF-κB诱饵ODNs处理可导致细胞增殖受到强烈抑制，尤其是在PC-3M细胞中。在FCM分析和AO/EB染色研究中观察到PC-3M细胞凋亡被诱导。在转染NF-κB诱饵的细胞中荧光素酶活性显著降低，但在转染对照诱饵的细胞中未降低。此外，我们发现一些内源性基因的表达降低，而其他基因转录本被诱导。EMSA证明NF-κB诱饵与NF-κB蛋白特异性结合。
这些发现表明，NF-κB激活通过调控靶基因表达在雄激素非依赖性前列腺癌的进展中起重要作用。因此，NF-κB抑制剂有望成为治疗雄激素非依赖性前列腺癌的一种治疗方法。NF-κB诱饵ODNs可能为人类恶性肿瘤开发治疗和研究工具。
