Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells.
Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells.
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抗增生因子降低了T24膀胱癌细胞中CKAP4的AKT磷酸化并改变基因表达。
DOI：
10.1186/1756-9966-29-160
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发表时间：
2010-12-10
期刊：
影响因子：
--
通讯作者：
Keay SK
中科院分区：
文献类型：
--
作者：
Shahjee HM;Koch KR;Guo L;Zhang CO;Keay SK
膀胱癌是世界范围内常见的恶性肿瘤，晚期膀胱癌患者的结局仍然很差。抗增殖因子（APF）是一种有效的上皮细胞增殖糖肽抑制剂，在间质性膀胱炎（一种膀胱上皮变薄和溃疡的疾病）患者的尿液中发现。APF通过细胞骨架相关蛋白4（CKAP 4）介导其在原代正常膀胱上皮细胞中的抗增殖活性。因为合成的无唾液酸-APF（as-APF）也已显示在体外以纳摩尔浓度抑制T24膀胱癌细胞增殖，并且因为APF的肽段与frizzled 8的部分100%同源，所以我们确定CKAP 4是否介导as-APF抑制T24细胞中的增殖和/或下游Wnt/frizzled信号传导事件。用针对CKAP 4的双链siRNA转染T24细胞，并用合成的as-APF或无活性对照肽处理;未经历电穿孔的细胞和用非靶（乱序）双链siRNA转染的细胞用作阴性对照。细胞增殖通过3 H-胸苷掺入测定。通过定量逆转录聚合酶链反应（qRT-PCR）测定Akt、糖原合成酶激酶3β（GSK 3 β）、β-连环蛋白、p53和基质金属蛋白酶2（MMP 2）mRNA的表达。蛋白质印迹法测定Akt、GSK-3β、MMP 2、β-catenin和p53蛋白表达以及Akt、GSK-3β和β-catenin磷酸化。APF可降低T24细胞增殖、MMP 2表达、Akt ser 473和thr 308磷酸化、GSK 3 β tyr 216磷酸化和β-catenin ser 45/thr 41磷酸化，而APF可增加非电穿孔和非靶siRNA转染细胞中p53表达和β-catenin ser 33、37/thr 41磷酸化。在这些细胞中，Akt、GSK 3 β或β-catenin的mRNA和总蛋白表达均未因APF而改变。此外，细胞增殖、MMP 2/p53 mRNA和蛋白表达以及Akt/GSK 3 β/β-catenin磷酸化对APF处理的响应的变化在CKAP 4 siRNA敲低后均被特异性消除。合成的as-APF通过CKAP 4受体抑制T24膀胱癌细胞的细胞增殖这种抑制作用的机制涉及调节特定细胞信号分子（Akt、GSK 3 β和β-连环蛋白）的磷酸化以及p53和MMP 2的mRNA和蛋白表达。
Urinary bladder cancer is a common malignancy worldwide, and outcomes for patients with advanced bladder cancer remain poor. Antiproliferative factor (APF) is a potent glycopeptide inhibitor of epithelial cell proliferation that was discovered in the urine of patients with interstitial cystitis, a disorder with bladder epithelial thinning and ulceration. APF mediates its antiproliferative activity in primary normal bladder epithelial cells via cytoskeletal associated protein 4 (CKAP4). Because synthetic asialo-APF (as-APF) has also been shown to inhibit T24 bladder cancer cell proliferation at nanomolar concentrations in vitro, and because the peptide segment of APF is 100% homologous to part of frizzled 8, we determined whether CKAP4 mediates as-APF inhibition of proliferation and/or downstream Wnt/frizzled signaling events in T24 cells. T24 cells were transfected with double-stranded siRNAs against CKAP4 and treated with synthetic as-APF or inactive control peptide; cells that did not undergo electroporation and cells transfected with non-target (scrambled) double-stranded siRNA served as negative controls. Cell proliferation was determined by 3H-thymidine incorporation. Expression of Akt, glycogen synthase kinase 3β (GSK3β), β-catenin, p53, and matrix metalloproteinase 2 (MMP2) mRNA was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Akt, GSK-3β, MMP2, β-catenin, and p53 protein expression, plus Akt, GSK-3β, and β-catenin phosphorylation, were determined by Western blot. T24 cell proliferation, MMP2 expression, Akt ser473 and thr308 phosphorylation, GSK3β tyr216 phosphorylation, and β-catenin ser45/thr41 phosphorylation were all decreased by APF, whereas p53 expression, and β-catenin ser33,37/thr41 phosphorylation, were increased by APF treatment in non-electroporated and non-target siRNA-transfected cells. Neither mRNA nor total protein expression of Akt, GSK3β, or β-catenin changed in response to APF in these cells. In addition, the changes in cell proliferation, MMP2/p53 mRNA and protein expression, and Akt/GSK3β/β-catenin phosphorylation in response to APF treatment were all specifically abrogated following CKAP4 siRNA knockdown. Synthetic as-APF inhibits cell proliferation in T24 bladder carcinoma cells via the CKAP4 receptor. The mechanism for this inhibition involves regulating phosphorylation of specific cell signaling molecules (Akt, GSK3β, and β-catenin) plus mRNA and protein expression of p53 and MMP2.
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影响因子：
2.1
作者：
Keay, S;Zhang, CO;Chai, TC
通讯作者：
Chai, TC
影响因子：
4.6
作者：
Keay, S;Seillier-Moiseiwitsch, F;Zhang, JL
通讯作者：
Zhang, JL
影响因子：
6.6
作者：
Keay, S;Warren, JW;Whitmore, KE
通讯作者：
Whitmore, KE
DOI：
10.1073/pnas.0404509101
发表时间：
2004-08-10
影响因子：
11.1
作者：
Keay, SK;Szekely, Z;Michejda, CJ
通讯作者：
Michejda, CJ
影响因子：
2.1
作者：
Gerhards, S;Jung, K;Loening, SA
通讯作者：
Loening, SA