www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 16 The roles of AKR1C1 and AKR1C2 in ethyl-3,4-dihydroxybenzoate induced esophageal squamous cell carcinoma cell death Wei Li1,*, Guixue Hou2,3,4,*, Dianrong Zhou5, Xiaomin Lou2, Yang Xu1, Siqi Liu2,4, Xiaohang Zhao1 1 State Key Laboratory of Molecular Oncology, Cancer Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China 2 CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China 3University of Chinese Academy of Sciences, Beijing, China 4Proteomics Division, BGI-Shenzhen, Shenzhen, Guangdong, China 5Third School of Clinical Medicine, Southern Medical University, Guangzhou, China *These authors have contributed equally to this work Correspondence to: Xiaohang Zhao, e-mail: [email protected] Siqi Liu, e-mail: [email protected] Yang Xu, e-mail: [email protected] Keywords: aldo-keto reductase 1C1/C2, ethyl-3, 4-dihydroxybenzoate, MRM, ESCC Received: November 19, 2015 Accepted: February 20, 2016 Published: February 27, 2016 ABSTRACT The aldo-keto reductase (AKR) superfamily of enzymes is critical for the detoxification of drugs and toxins in the human body; these enzymes are involved not only in the development of drug resistance in cancer cells but also in the metabolism of polycyclic aromatic hydrocarbons. Here, we demonstrated that AKR1C1/C2 increased the metabolism of ethyl-3,4-dihydroxybenzoate (EDHB) in esophageal squamous cell carcinoma (ESCC) cells. Previous studies have shown that EDHB can effectively induce esophageal cancer cell autophagy and apoptosis, and the AKR1C family represents one set of highly expressed genes after EDHB treatment. To explore the cytotoxic effects of EDHB, esophageal cancer cells with higher (KYSE180) or lower (KYSE510) AKR1C expression levels were evaluated in this study. The proliferation of KYSE180 cells was inhibited more effectively than that of KYSE510 cells by EDHB treatment. Furthermore, the effective subunits of the AKR superfamily, AKR1C1/C2, were quantitatively identified using multiple reaction monitoring (MRM) assays. The sensitivity of esophageal cancer cells to EDHB was significantly attenuated by the siRNA knockdown of AKR1C1/C2. Moreover, the expression of autophagy inducers (Beclin, LC3II and BNIP3) and NDRG1 was significantly elevated in KYSE180 cells, but not in KYSE510 cells, after EDHB treatment. When autophagy was inhibited by 3-methyladenine, KYSE180 cells exhibited an increased sensitivity to EDHB, which may be a metabolic substrate of AKR1C1/C2. These results indicated that ESCC patients with high AKR1C1/C2 expression may be more sensitive to EDHB, and AKR1C1/ C2 may facilitate EDHB-induced autophagy and apoptosis, thus providing potential guidance for the chemoprevention of ESCC. INTRODUCTION cases in Asian countries [2]. Currently, the precise etiology and pathogenesis of esophageal cancer are unclear, and Esophageal cancer is the eighth most common there is a lack of effective methods for early diagnosis. As cancer and the sixth leading cause of cancer-related death a consequence, the majority of esophageal cancer patients worldwide [1]. Esophageal squamous cell carcinoma are diagnosed at intermediate and advanced stages; their (ESCC) accounts for nearly 90% of esophageal cancer therapeutic results are poor [3, 4], and the 5-year survival www.impactjournals.com/oncotarget 21542 Oncotarget rate is no greater than 19% [5, 6]. The development and metabolic substrate of AKR1C. The most recent study on progression of esophageal cancer are multi-factorial, this topic showed that prostate tumor samples with activated multi-stage progressive processes that result from the joint AKR1C1, an androgen-metabolizing enzyme, harbor high action of environmental and genetic factors. However, Sp1 and c-FLIP expression and low AKR1C1, ERβ and interventions that treat precancerous lesions could reverse Sp3 expression. AKR1C1/ERβ activation induces apoptosis the evolution of esophageal cancer. Phenolic compounds, by downregulating c-FLIP expression, providing a new such as ethyl-3,4-dihydroxybenzoate (EDHB), form a approach for the treatment of prostate cancer [17]. Our results class of chemical anticancer agents that act on the Nrf2/ showed that an esophageal cancer cell line overexpressing ARE pathway. Such compounds are widely distributed in AKR1C was more sensitive to EDHB-induced cell death. fruits and vegetables and show strong antioxidant effects. However, in the AKR1C subfamily, AKR1C1/C2–AKR1C4 EDHB is also known as protocatechuic acid ethyl share a high degree of homology. In particular, the homology ester. The monomer component of EDHB is present in a between AKR1C1 and AKR1C2 is 98%, with only seven variety of plant species, mainly in the leaves and roots. different amino acids. To analyze which AKR1C protein EDHB was identified in studies on the composition of increases EDHB-induced cell death in ESCC, we utilized the peanut seed coat, wine, and tea leaves, and it exerts a quantitative method, mTRAQ-based multiple reaction relatively low antioxidant activity [7–9]. Therefore, EDHB monitoring (MRM), to measure human AKR1C levels after can be used as a fat and cream antioxidant, a food additive, EDHB treatment. and a pharmaceutical intermediate. EDHB is a substrate When combined with the newly released mTRAQ analog of prolyl hydroxylase and acts as a competitive reagent, a non-isobaric variant of the iTRAQ tag that is inhibitor. It inhibits the ubiquitination/degradation available in two versions, the MRM approach enables the of hypoxia inducible factor (HIF) and promotes the absolute quantification of peptides and proteins via isotope- expression of downstream genes (e.g., erythropoietin and dilution mass spectrometry. The method is conceptually vascular endothelial growth factor), thus exerting critical similar to Western blotting but differs substantially in biological effects [10]. EDHB also protects L6 myoblasts implementation, assay reliability, and the quality of the results. from hypoxia-induced oxidative damage [11] and inhibits A Western blot essentially depends on antibody specificity. collagen secretion from breast cancer cells, thus preventing MRM is a better alternative for quantifying protein abundance tumor metastasis. This activity has been shown to reduce using liquid chromatography (LC) or SDS-PAGE to separate tumor fibrosis and metastasis in a mouse model of breast proteins, especially when a suitable antibody is unavailable cancer, thereby providing a new potential treatment for and highly precise quantification is necessary; Zhang et al. breast cancer [12]. Previous research has also shown employed this method to quantitate the levels of AKR1A1, that EDHB inhibits esophageal cancer cell proliferation AKR1C1/C2, AKR1C3, AKR1C4, AKR1B1, and AKR1B10 by increasing autophagy and apoptosis through NDRG1 in seven different cancer cell lines and to ascertain the absolute and BNIP3 and markedly increases AKR1C1, AKR1C2, quantities of all proteins in each cell line [18–19]. In MRM, and AKR1C3 mRNA expression [13]. Therefore, it may the mass spectrometer (MS) is set to monitor only specific be possible to identify new methods to inhibit esophageal mass/charge (m/z) values; as a consequence, the probability of cancer by investigating the effect of EDHB on esophageal detecting low levels of a peptide as part of a complex mixture cancer cells and its underlying molecular mechanism. is much higher [20]. Thus, MRM has been the method of The members of the aldo-keto reductase (AKR) choice for the quantification of similar proteins, especially superfamily are monomeric cytoplasmic proteins with a when a suitable antibody is not available. molecular weight of 34–37 kDa. AKRs catalyze the reduction This study utilized an MRM assay to confirm of aldehydes and ketones into the corresponding alcohol that AKR1C1/C2 are involved in the EDHB-induced compounds, and reduced nicotinamide adenine dinucleotide inhibition of esophageal cancer cell proliferation. EDHB phosphate (NADPH) serves as a coenzyme. The AKRs was used as a substrate of AKR1C1/C2, and KYSE 180 can be divided into 15 families, and the AKR1C subfamily cells overexpressing AKR1C1/C2 were more sensitive to includes four isoenzymes, AKR1C1–AKR1C4. These EDHB, which potentially provides guidance for the use of isoenzymes are capable of reducing 3-, 17-, and 20-ketone EDHB for the treatment of esophageal cancer. steroids in vitro [14] and of polycyclic aromatic hydrocarbons (PAHs) [15]. Additionally, AKR1C participates in the RESULTS metabolism of numerous exogenous drugs and enhances the metabolism of certain endogenous and exogenous substrates. High levels of AKR1C enhanced the sensitivity of Many important drugs, such as doxorubicin, daunorubicin, ESCC cells to EDHB haloperidol, and the novel anticancer drug oracin, are metabolized by carbonyl-reducing enzymes, including To analyze AKR1C expression in different ESCC AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, and cells, total protein was extracted from KYSE 30, KYSE 140, AKR1C3 [16]. However, EDHB is an exogenous substance KYSE 150, KYSE 170, KYSE 180, KYSE 410 and KYSE with a similar structure to the metabolic substrates of AKR1C 510 cells. Western blot analysis showed that AKR1C was (i.e., PAHs). For this reason, we confirmed that EDHB is a expressed at higher (KYSE 180) or lower (KYSE 510) levels www.impactjournals.com/oncotarget 21543 Oncotarget in certain ESCC cell lines (Figure 1A).