PPARgamma-mediated ALDH1A3 suppression exerts anti-proliferative effects in lung cancer by inducing lipid peroxidation

Context: The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear.
Objectives: To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth.
Materials and methods:In silico analysis using microarray dataset was performed to screen the positive correlation between PPARγ and all ALDH isoforms. NUBIscan software and ChIP assay were used to identify the binding sites (BSs) of PPARγ on ALDH1A3 promoter. The expression of ALDH1A3 under thiazolidinedione (TZD) treatment was evaluated by QPCR and Western Blot in HBEC and H1993 cell lines. Upon treatment of TZD, colony formation assay was used to check cell growth inhibition and 4-hydroxy-2-nonenal (4HNE) production as lipid peroxidation marker was determined by Western Blot in PPARγ positive cell H1993 and PPARγ negative cell H1299.
Results: Compared to other ALDH isoforms, ALDH1A3 showed the highest positive correlation to PPARγ expression. ALDH1A3 upregulated PPARγ expression while PPARγ activation suppressed ALDH1A3. Among 2 potential screened PPARγ response elements, BS 1 and 2 in the promoter of ALDH1A3 gene, PPARγ bound directly to BS2. Ligand activation of PPARγ suppressed mRNA and protein expression of ALDH1A3. Growth inhibition was observed in H1993 (PPARγ positive cell) treated with PPARγ activator and ALDH inhibitor compared to H1299 (PPARγ negative cell). PPARγ activation increased 4HNE which is known to be suppressed by ALDH1A3.
Conclusions: ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.

研究背景：过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma, PPARγ）在肺癌中的代谢功能尚不明确。
研究目的：旨在明确PPARγ与ALDH1A3诱导的脂质过氧化在抑制肺癌细胞生长过程中的关联。
材料与方法：通过芯片数据集开展in silico生物信息学分析，筛选PPARγ与所有醛脱氢酶（aldehyde dehydrogenase, ALDH）同工型之间的正相关关联；采用NUBIscan软件与染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验鉴定PPARγ在ALDH1A3基因启动子区域的结合位点（binding sites, BSs）；通过实时定量聚合酶链反应（quantitative polymerase chain reaction, QPCR）与蛋白质印迹（Western Blot）检测噻唑烷二酮（thiazolidinedione, TZD）处理后，HBEC与H1993细胞系中ALDH1A3的表达水平；在PPARγ阳性细胞系H1993与PPARγ阴性细胞系H1299中，经TZD处理后，采用集落形成实验检测细胞生长抑制情况，并通过蛋白质印迹检测作为脂质过氧化标志物的4-羟基-2-壬烯醛（4-hydroxy-2-nonenal, 4HNE）的产生水平。
研究结果：与其他ALDH同工型相比，ALDH1A3与PPARγ的表达呈现最强的正相关性；ALDH1A3可上调PPARγ的表达，而PPARγ活化则会抑制ALDH1A3的表达；在筛选得到的2个潜在PPARγ应答元件中，ALDH1A3基因启动子区域的结合位点1与结合位点2内，PPARγ可直接结合结合位点2；PPARγ的配体活化可抑制ALDH1A3的mRNA与蛋白表达水平；与PPARγ阴性细胞系H1299相比，经PPARγ激活剂与ALDH抑制剂共同处理的H1993细胞出现了生长抑制现象；PPARγ活化可提升4HNE的水平，而已知ALDH1A3可抑制4HNE的产生。
研究结论：ALDH1A3的抑制可能是PPARγ发挥肿瘤抑制功能的途径之一；本研究有助于进一步阐明PPARγ在肺癌中的作用机制。