Zheng Qiaoping, Gan Guifang, Gao Xianfu, Luo Qingqiong, Chen Fuxiang
Department of Clinical Immunology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, People's Republic of China.
Shanghai Profleader Biotech Co., Ltd., Shanghai, People's Republic of China.
Onco Targets Ther. 2021 Mar 4;14:1673-1687. doi: 10.2147/OTT.S288692. eCollection 2021.
Indolamine 2,3-dioxygenase (IDO) is the rate limiting enzyme of tryptophan degradation and is a negative prognostic factor in oral squamous cell carcinoma (OSCC) patients, while the underlying molecular mechanism remains unclear. This research aimed to explore the IDO expression and its biological functions in OSCC.
IDO expression was analyzed by qPCR, Western blots, and immunohistochemistry (IHC) in OSCC cell lines and tissue specimens. Tryptophan and kynurenine content were determined by UPLC-MS/MS in serum samples of OSCC patients and healthy controls. Oncomine databases and Kaplan-Meier survival analyses were used to identify the IDO expression and its correlation with OSCC prognosis. Cell counting, CCK8 assay, flow cytometry, cell cycle, and EdU incorporation assays were used to assess the effect of IDO inhibition on OSCC growth either by shRNA or the IDO-specific inhibitor (epacadostat) in vitro. An OSCC xenograft mouse model was established to verify the predicted function of IDO inhibition in vivo. Mechanistically, an 84-gene apoptosis PCR array and rescue experiment were used to characterize the underlying mechanism involved in IDO-regulated apoptosis in OSCC.
IDO expression was upregulated in OSCC cell lines and tissues and was negatively correlated with OSCC progression. Lentivirus-mediated IDO knockdown and epacadostat significantly reduced viability and promoted apoptosis of OSCC cells in vitro and in vivo. The apoptosis PCR array identified BCL2 related protein A1 (BCL2A1) as the most obviously changed gene at the transcriptional level. IDO inhibition downregulated BCL2A1 expression, increased the expression and translocation of cytochrome c, thus promoted apoptosis in OSCC. Overexpression of BCL2A1 reversed the pro-apoptotic effect of IDO inhibition.
The present results revealed that IDO directly affect the growth of OSCC cells by regulating BCL2A1 expression. IDO and the IDO-BCL2A1-cytochrome c axis may be potential therapeutic targets for OSCC.
吲哚胺2,3-双加氧酶（IDO）是色氨酸降解的限速酶，是口腔鳞状细胞癌（OSCC）患者的负性预后因素，但其潜在分子机制仍不清楚。本研究旨在探讨IDO在OSCC中的表达及其生物学功能。
采用qPCR、蛋白质免疫印迹法和免疫组织化学（IHC）分析OSCC细胞系和组织标本中IDO的表达。采用超高效液相色谱-串联质谱法（UPLC-MS/MS）测定OSCC患者和健康对照者血清样本中的色氨酸和犬尿氨酸含量。利用Oncomine数据库和Kaplan-Meier生存分析确定IDO的表达及其与OSCC预后的相关性。采用细胞计数、CCK8检测、流式细胞术、细胞周期和EdU掺入检测等方法，在体外评估shRNA或IDO特异性抑制剂（依帕司他）抑制IDO对OSCC生长的影响。建立OSCC异种移植小鼠模型，在体内验证IDO抑制的预测功能。机制上，采用84基因凋亡PCR阵列和挽救实验来表征IDO调节OSCC细胞凋亡的潜在机制。
IDO在OSCC细胞系和组织中表达上调，与OSCC进展呈负相关。慢病毒介导的IDO敲低和依帕司他显著降低了OSCC细胞在体外和体内的活力并促进其凋亡。凋亡PCR阵列确定BCL2相关蛋白A1（BCL2A1）是转录水平上变化最明显的基因。IDO抑制下调BCL2A1表达，增加细胞色素c的表达和转位，从而促进OSCC细胞凋亡。BCL2A1的过表达逆转了IDO抑制的促凋亡作用。
本研究结果表明，IDO通过调节BCL2A1表达直接影响OSCC细胞的生长。IDO及IDO-BCL2A1-细胞色素c轴可能是OSCC的潜在治疗靶点。
