Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells.
Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells.
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DOI：
10.1016/j.yexcr.2012.03.033
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发表时间：
2012-08-01
影响因子：
3.7
通讯作者：
Ray, Swapan K.
中科院分区：
文献类型：
--
作者：
Hossain, Md Motarab;Banik, Naren L.;Ray, Swapan K.
神经母细胞瘤是一种主要发生在儿童的实体瘤。恶性神经母细胞瘤预后差，因为常规化疗药物几乎没有效果。Survivin在某些恶性神经母细胞瘤中高表达，在抑制分化和凋亡、促进细胞增殖、侵袭和血管生成等方面发挥重要作用。我们研究了在恶性神经母细胞瘤细胞中，通过生存素短发夹RNA（shRNA）质粒敲低生存素，然后用（-）-表没食子儿茶素-3-没食子酸酯（EGCG）（一种绿色茶类黄酮）治疗的结果。Western blotting和激光扫描共聚焦免疫荧光显微镜显示Survivin在恶性神经母细胞瘤SK-N-BE 2和SH-SY 5 Y细胞株中呈高表达，在SK-N-DZ细胞株中呈弱表达。Survivin在恶性神经母细胞瘤IMR 32细胞系中表达微弱。我们用Survivin shRNA转染SK-N-BE 2和SH-SY-5 Y细胞，用EGCG处理，并证实Survivin在mRNA和蛋白水平上的敲低。生存素敲低诱导神经元分化的形态学特征，因为我们观察到以下原位亚甲蓝染色。Survivin shRNA和EGCG的组合通过增加NFP、NSE和e-cadherin的表达以及降低Notch-1、ID 2、hTERT和PCNA的表达来生物化学地促进神经元分化。我们的原位Wright染色和Annexin V-FITC/PI染色显示，联合治疗分别在诱导凋亡的形态学和生化特征方面非常有效。凋亡发生与caspase-8的激活和裂解的Bid到tBid，增加Bax：Bcl-2的比例，线粒体释放细胞色素c，并增加钙蛋白酶和caspase-3的表达和活性。联合治疗减少了细胞通过基质胶的迁移，并抑制了增殖因子（p-Akt和NF-κB）、侵袭因子（MMP-2和MMP-9）和血管生成因子（VEGF和b-FGF）。此外，在体外细胞的网络形成能力显着抑制生存素沉默和完全由生存素沉默和EGCG处理的组合。总的来说，生存素沉默增强了EGCG在具有生存素过表达的人恶性神经母细胞瘤细胞中的抗癌作用。
Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (−)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in expression of NFP, NSE, and e-cadherin and also decreases in expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression.
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影响因子：
8.8
作者：
Jung, Y D;Kim, M S;Shin, B A;Chay, K O;Ahn, B W;Liu, W;Bucana, C D;Gallick, G E;Ellis, L M
通讯作者：
Ellis, L M
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影响因子：
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作者：
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通讯作者：
Altieri, Dario C.
影响因子：
5.8
作者：
Janardhanan, Rajiv;Banik, Naren L.;Ray, Swapan K.
通讯作者：
Ray, Swapan K.