Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.
J Exp Clin Cancer Res. 2010 Dec 10;29(1):160. doi: 10.1186/1756-9966-29-160.
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.
膀胱癌是一种常见的恶性肿瘤，晚期膀胱癌患者的预后仍然较差。抗增殖因子（APF）是一种强效的上皮细胞增殖糖肽抑制剂，最初在间质性膀胱炎患者的尿液中发现，间质性膀胱炎是一种膀胱上皮变薄和溃疡的疾病。APF 通过细胞骨架相关蛋白 4（CKAP4）在原代正常膀胱上皮细胞中发挥其抗增殖活性。因为合成去唾液酸-APF（as-APF）也已被证明可以在体外以纳摩尔浓度抑制 T24 膀胱癌细胞的增殖，并且因为 APF 的肽段与 frizzled 8 的一部分 100%同源，所以我们确定 CKAP4 是否介导 as-APF 抑制 T24 细胞的增殖和/或下游 Wnt/frizzled 信号事件。
用针对 CKAP4 的双链 siRNA 转染 T24 细胞，并用合成的 as-APF 或非活性对照肽处理；未进行电穿孔的细胞和转染非靶向（乱序）双链 siRNA 的细胞作为阴性对照。通过 3H-胸苷掺入测定细胞增殖。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（GSK3β）、β-连环蛋白、p53 和基质金属蛋白酶 2（MMP2）mRNA 的表达。通过 Western blot 测定 Akt、GSK-3β、MMP2、β-连环蛋白和 p53 蛋白表达以及 Akt、GSK-3β 和 β-连环蛋白磷酸化。
APF 降低了 T24 细胞的增殖、MMP2 表达、Akt ser473 和 thr308 磷酸化、GSK3β tyr216 磷酸化和β-连环蛋白 ser45/thr41 磷酸化，而 APF 处理非电穿孔和非靶向 siRNA 转染细胞增加了 p53 表达和β-连环蛋白 ser33、37/thr41 磷酸化。在这些细胞中，APF 既不改变 Akt、GSK3β 或β-连环蛋白的 mRNA 也不改变其总蛋白表达。此外，APF 处理后，细胞增殖、MMP2/p53 mRNA 和蛋白表达以及 Akt/GSK3β/β-连环蛋白磷酸化的变化均在 CKAP4 siRNA 敲低后特异性消除。
合成的 as-APF 通过 CKAP4 受体抑制 T24 膀胱癌细胞的增殖。这种抑制的机制涉及调节特定细胞信号分子（Akt、GSK3β 和β-连环蛋白）的磷酸化以及 p53 和 MMP2 的 mRNA 和蛋白表达。
