Study for a tumor suppressive serpin, maspin in cervical cancer

我们最近的一系列研究表明，正常和恶性宫颈鳞状上皮组织中肿瘤相关丝氨酸蛋白酶抑制物(SERPIN)、鳞状细胞癌(SCC)抗原的分子异质性存在明显差异。另一方面，尽管Maspin是乳腺癌中一种42 kDa的独特抑癌丝氨酸，在包括肿瘤进展、氧化应激等生理病理条件下与多种靶分子相互作用，具有多方面的生物学功能，但目前尚不清楚翻译后修饰对Maspin与靶分子如谷胱甘肽S转移酶(GST)的特异性结合作用。因此，本研究的目的是利用不同的电泳法分析maspin的分子异质性，并在已知表达丰富的maspin蛋白的MCF-10A细胞中鉴定其修饰。我们成功地鉴定了氧化应激下通过分子内二硫键连接产生的内源性maspin的新形式。此外，谷胱甘肽小球下拉实验表明，分子内二硫键结合的maspin失去了与内源GST的结合活性，这表明分子内二硫键结合的maspin在氧化应激下可能具有一些独特的特性，尽管这种修饰的确切生物学意义仍然不清楚。对宫颈癌原发灶组织进行的Western印迹分析表明，有盆腔淋巴结转移的宫颈癌患者的maspin表达水平较无盆腔淋巴结转移的宫颈癌患者低，提示抑癌基因maspin的抑制或翻译后修饰可能与宫颈癌的进展有关。

Our series of recent study has demonstrated that there is a distinct difference between normal and malignant uterine cervical squamous epithelial tissues in the molecular heterogeneity of the tumor-related serine proteinase inhibitor (serpin), Squamous Cell Carcinoma (SCC) antigen. On the other hand, although maspin, identified as a 42-kDa unique tumor suppressive serpin in breast cancer, has multifaceted biological functions by interacting with various target molecules under physiological and pathological conditions including tumor progression, oxidative stress and so on, it is unknown what post-translational modification contributes the specific binding affinity of maspin to target molecules, such as glutathione S-transferase (GST). The aim of this study is therefore to analyze the molecular heterogeneity of maspin and identify its modification in MCF-10A cell, which is known to express abundant maspin protein, using various electrophoretic analyses. We successfully identified the novel form of endogenous maspin generated by intramolecular disulfide-bonded linkage under oxidative stress. Furthermore, glutathione beads pull-down assay revealed that intramolecular disulfide-bonded maspin lost the binding activity with endogenous GST, indicating that intramolecular disulfide-bonded maspin might have some distinct properties under oxidative stress, although the precise biological significance of this modification remains elusive. The western blot analysis using cervical primary cancer tissues showed that maspin expression levels in cervical cancer patients with pelvic lymph node metastasis were relative low compared to those with no pelvic lymph node metastases, suggesting that the suppression or post-translational modification of the tumor suppressive serpin, maspin might be related to tumor progression in cervical cancer.