Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, China.
Cancer Biother Radiopharm. 2011 Oct;26(5):647-55. doi: 10.1089/cbr.2011.0984. Epub 2011 Sep 8.
The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, an mda-7/IL-24 mutant, RGD-mda-7, which includes the cell adhesive sequence 164Arg-165Gly-166Asp (RGD motif), was constructed and evaluated for bioactivity. RGD peptide binds to integrins α(V)β(3) and α(V)β(5), which are selectively expressed in tumor neovasculature and in the surface of some tumor cells. The wtmda-7/IL-24 and RGD-mda-7 were expressed in Escherichia coli and then purified and renatured. The immunostimulatory activity of RGD-mda-7 was assayed by stimulating peripheral blood mononuclear cells. The results suggested that the abilities of RGD-mda-7 to induce IL-6, TNF-α, and IFN-γ production were higher than wtmda-7/IL-24. Tumor targeting of RGD-mda-7 was assayed using cell adhesion experiments. The antitumor effect of the purified RGD-mda-7 on cell proliferation in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake, cell apoptosis by staining with fluorescent probes of FITC-annexin V and DAPI, and caspase-3 expression and activity. The in vitro results showed that RGD-mda-7 inhibited the proliferation of multiple tumor cell lines (Hela, ACHN, HepG2, and A549). Staining with fluorescent probes of FITC-annexin V and DAPI indicated that RGD-mda-7 could induce apoptosis more effectively in four tumor cell lines than wtmda-7/IL-24, but has no effect on normal cell line NHLF. Western blotting showed that treatment of tumor cells with RGD-mda-7 could activate apoptotic pathway by cleavage of caspase-3 as same as wtmda-7/IL-24. Further, RGD-mda-7 group showed a higher cleaved level of caspase-3, but not in NHLF cells. These results demonstrate that RGD-MDA-7 possesses more potent antitumor effects than wtmda-7/IL-24 and therefore merits further investigation in preclinical and clinical studies.
黑色素瘤分化相关基因-7/白细胞介素-24 基因（mda-7/IL-24）是一种新型的肿瘤抑制因子/细胞因子基因，具有强大的肿瘤抑制活性，而不会损伤正常细胞。为了增强抗肿瘤效果，构建并评估了一种 mda-7/IL-24 突变体 RGD-mda-7，其包含细胞黏附序列 164Arg-165Gly-166Asp（RGD 基序）。RGD 肽与整合素 α(V)β(3)和 α(V)β(5)结合，这些整合素在肿瘤新生血管和一些肿瘤细胞表面选择性表达。wtmda-7/IL-24 和 RGD-mda-7 在大肠杆菌中表达，然后进行纯化和复性。通过刺激外周血单个核细胞来检测 RGD-mda-7 的免疫刺激活性。结果表明，RGD-mda-7 诱导 IL-6、TNF-α 和 IFN-γ 产生的能力高于 wtmda-7/IL-24。通过细胞黏附实验检测 RGD-mda-7 的肿瘤靶向性。使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）摄取评估纯化的 RGD-mda-7 对体外细胞增殖的抗肿瘤作用，通过 FITC-annexin V 和 DAPI 荧光探针染色评估细胞凋亡，以及 caspase-3 表达和活性。体外结果表明，RGD-mda-7 抑制多种肿瘤细胞系（Hela、ACHN、HepG2 和 A549）的增殖。FITC-annexin V 和 DAPI 荧光探针染色表明，与 wtmda-7/IL-24 相比，RGD-mda-7 能更有效地诱导四种肿瘤细胞系的凋亡，但对正常细胞系 NHLF 无影响。Western blot 表明，用 RGD-mda-7 处理肿瘤细胞可通过切割 caspase-3 激活凋亡途径，与 wtmda-7/IL-24 相同。此外，RGD-mda-7 组 caspase-3 的切割水平更高，但在 NHLF 细胞中没有。这些结果表明，RGD-MDA-7 比 wtmda-7/IL-24 具有更强的抗肿瘤作用，因此值得进一步在临床前和临床研究中进行研究。
