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

<b>Context:</b> The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear. <b>Objectives:</b> To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth. <b>Materials and methods:</b><i>In silico</i> 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. <b>Results:</b> 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 <i>ALDH1A3</i> 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. <b>Conclusions:</b> ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.

<b>研究背景：</b>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目前仍不明确。<b>研究目的：</b>本研究旨在明确PPARγ与ALDH1A3诱导的脂质过氧化之间的关联，及其对肺癌细胞生长的抑制作用。<b>材料与方法：</b>通过芯片数据集开展in silico分析，筛选PPARγ与所有醛脱氢酶（aldehyde dehydrogenase, ALDH）同工型的正相关关联。使用NUBIscan软件及染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验，鉴定PPARγ在ALDH1A3启动子区域的结合位点（binding sites, BSs）。采用实时定量聚合酶链式反应（quantitative polymerase chain reaction, QPCR）与蛋白质免疫印迹（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。分别以PPARγ阳性细胞系H1993及PPARγ阴性细胞系H1299为研究对象，经TZD处理后，通过克隆形成实验检测细胞生长抑制情况，并通过Western Blot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。<b>研究结果：</b>相较于其他ALDH同工型，ALDH1A3与PPARγ表达的正相关程度最高。ALDH1A3可上调PPARγ的表达水平，而PPARγ激活则会抑制ALDH1A3的表达。在筛选得到的2个潜在PPARγ应答元件中，即ALDH1A3基因启动子区域的BS1与BS2位点，PPARγ可直接结合至BS2位点。PPARγ的配体激活可下调ALDH1A3的mRNA及蛋白表达水平。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（PPARγ阳性细胞）出现了显著的生长抑制现象。PPARγ激活可升高4HNE的水平，而4HNE的表达可被ALDH1A3所抑制。<b>研究结论：</b>ALDH1A3的抑制可能是PPARγ发挥肿瘤抑制功能的途径之一。本研究为阐明PPARγ在肺癌中的作用提供了更为全面的理论依据。