van de Schootbrugge Chantal, Schults Elisabeth M J, Bussink Johan, Span Paul N, Grénman Reidar, Pruijn Ger J M, Kaanders Johannes H A M, Boelens Wilbert C
Department of Biomolecular Chemistry, Institute for Molecules and Materials and Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, 271, RIMLS, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
BMC Cancer. 2014 Apr 11;14:252. doi: 10.1186/1471-2407-14-252.
The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. αB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether αB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces αB-crystallin expression in vitro and in this way may confer hypoxic cell survival.
In 38 HNSCC biopsies, the overlap between immunohistochemically stained αB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of αB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of αB-crystallin on cell survival under hypoxic conditions.
In all biopsies αB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased αB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, αB-crystallin expression was increased. αB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced αB-crystallin upregulation. Moreover, it was found that decreased αB-crystallin levels reduced cell survival under hypoxic conditions.
We provide the first evidence that hypoxia stimulates upregulation of αB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of αB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.
头颈部鳞状细胞癌（HNSCC）中缺氧的存在与治疗抵抗及转移形成风险增加相关。αB-晶状体蛋白（HspB5）是一种小热休克蛋白，也与HNSCC的转移形成有关。在本研究中，我们调查了αB-晶状体蛋白在HNSCC活检组织缺氧区域的蛋白表达是否增加，并分析了缺氧在体外是否诱导αB-晶状体蛋白表达，以及以此方式是否赋予缺氧细胞生存能力。
在38例HNSCC活检组织中，确定免疫组化染色的αB-晶状体蛋白与匹莫硝唑加合物（缺氧标志物）之间的重叠情况。此外，分析了HNSCC细胞系在缺氧和复氧条件下以及暴露于活性氧（ROS）和ROS清除剂N-乙酰半胱氨酸（NAC）后的αB-晶状体蛋白表达水平。使用小干扰RNA（siRNA）介导的敲低来确定αB-晶状体蛋白对缺氧条件下细胞生存的影响。
在所有活检组织中，αB-晶状体蛋白在缺氧区域比在正常氧合区域更丰富。值得注意的是，缺氧降低了HNSCC细胞系中αB-晶状体蛋白的mRNA表达。仅在复氧后，即刺激ROS形成的条件下，αB-晶状体蛋白表达增加。细胞外ROS也增加了αB-晶状体蛋白的mRNA水平，并且NAC消除了复氧诱导的αB-晶状体蛋白上调。此外，发现降低αB-晶状体蛋白水平会降低缺氧条件下的细胞生存能力。
我们提供了首个证据，证明缺氧刺激HNSCC中αB-晶状体蛋白的上调。这种上调不是由低氧压力引起的，而更可能是由ROS形成引起的。αB-晶状体蛋白的较高表达可能导致这些细胞在缺氧条件下的存活时间延长。
