Background
Gastric cancer (GC) is one of the most common malignant tumours in the world with morbidity and mortality accounting for the fourth and second places among malignant tumours. Each year, more than 800,000 new patients are diagnosed with GC, of which nearly 90% have advanced GC, and few patients are eligible for surgery. Because of the heterogeneity of GC, the efficacy of traditional radiotherapies and chemotherapies is not satisfactory. In recent years, biotherapy and targeted therapy for GC have made great progress, but the prognosis of patients with GC is still not optimistic, and the molecular mechanisms of GC occurrence and development are still unclear [
1]. Autophagy is a common physiological process in normal and GC cells. Abnormal levels of autophagy have major effects on the occurrence and progression of GC. Therefore, elucidating the mechanism of autophagy in the development of GC has great clinical significance.
Reactive oxygen species (ROS) are important signalling molecules in cells, which participate in the transmission of information via multiple signalling pathways [
2,
3]. Excessive ROS induce tumour cell autophagy and apoptosis by inhibiting PI3K/Akt and other pathways, thereby inhibiting the occurrence and development of tumours [
4]. For example, salinomycin promotes autophagy and apoptosis of prostate cancer cells through PI3K/Akt/mTOR and ERK/p38 MAPK pathways by increasing the cellular ROS level [
5]. Inhibiting the autophagy level of prostate cancer cells increases their apoptosis level induced by salinomycin, thereby increasing the chemotherapy sensitivity of salinomycin. Ciclopirox olamine increases ROS levels in rectal cancer cells by affecting mitochondrial functions and then induces apoptosis and protective autophagy through the AMPK pathway [
6]. Inhibiting this cytoprotective autophagy increases the level of apoptosis in rectal cancer cells induced by ciclopirox olamine. ROS in tumour cells are not only produced by stimulation from the external environment, but are also generated by the cell itself or as a byproduct of other biological reactions [
7‐
9]. Post-translational modifications of proteins, such as ubiquitination and phosphorylation, play an important role in this process. For example, changes in the level of nuclear factor erythroid 2-related factor 2 (Nrf2) SUMOylation affect the intracellular ROS level and thus the autophagy level of cells, which ultimately affect the occurrence and development of hepatocellular carcinoma [
10].
BDH2 is a short-chain dehydrogenase/reductase family member originally named as DHRS6 [
11]. The human BDH2 gene is located on 4q and encodes 245 amino acids. BDH2 is widely expressed in the cytoplasm of epithelial cells of organs such as the kidney, small intestines, oral cavity, and breast. BDH2 may be a multifunctional gene in mammalian cells. It is a novel cytosolic-type 2-hydroxybutyrate dehydrogenase and has a physiological role in the use of cytosolic ketone bodies that can subsequently enter mitochondria and the tricarboxylic acid cycle [
12]. BDH2 also catalyses the synthesis of 2,5-dihydroxybenzoic acid during biosynthesis of enterobactin [
13]. In addition, BDH2 may be an independent poor prognosis marker of acute myeloid leukaemia by affecting apoptosis. BDH2 is regulated by long-chain non-coding RNA TP73-AS1, which affects oesophageal squamous cell carcinoma cell proliferation and apoptosis [
14]. However, the effect of BDH2 expression on autophagy of GC cells and its possible mechanism have not been reported.
In this study, we analysed the expression level of BDH2 in paired GC tissues by immunohistochemical analysis, and the correlation between BDH2 expression levels and clinicopathological features and prognosis of GC. We found that low expression of BDH2 is an independent molecular marker of a poor prognosis of GC patients. GC cell lines stably expressing BDH2 were constructed, and the effect of the change of the BDH2 expression level on the apoptosis and autophagy of GC cells was observed. On the molecular level, we demonstrated that BDH2 regulates the Keap1-Nrf2 interaction and promotes proteasomal degradation of Nrf2. Increasing the levels of ubiquitination/degradation of Nrf2 led to intracellular ROS accumulation, thereby altering the PI3K/Akt /mTOR pathway to inhibit the growth of GC cells in vivo and in vitro.
Materials and methods
Patients and tissue samples
We collected 171 paired tumour and adjacent non-cancerous gastric tissues from January 2010 to December 2010 at the Affiliated Hospital of Nantong University (Jiangsu, China). Detailed clinicopathological information is provided in Table
1. Another cohort of 30 fresh GC cases was collected from the Department of General Surgery for western blot analysis. All patients were definitively diagnosed with GC by pathological histology and had not received adjuvant chemotherapy, radiation therapy, or immunotherapy before surgical excision of tumours. All experiments were conducted with the approval of the Committee for the Ethical Review of Research involving Human Subjects at Nantong University Affiliated Hospital. Written informed consent was obtained from each patient before sample collection.
Table 1
Correlation between BDH2 expression in GC tissues and clinicopathological features of GC patients
Gender | | | | 0.371 |
Male | 111 | 80(72.1%) | 31(27.9%) | |
Female | 60 | 47(78.3%) | 13(21.7%) | |
Age (years) | | . | . | 0.433 |
≤60 | 73 | 52(71.2%) | 21(28.8%) | |
> 60 | 98 | 75(76.5%) | 23(23.5%) | |
Degree of differentiation | | | | 0.177 |
Well | 18 | 11(61.1%) | 7(38.9%) | |
Moderate/Poor | 153 | 116(75.8%) | 37(24.2%) | |
Tumor diameter (cm) | | | | 0.124 |
< 5 | 112 | 79(70.5%) | 33(29.5%) | |
≥5 | 59 | 48(81.4%) | 11(18.6%) | |
Tumor localization | | | | 0.536 |
Up | 19 | 13(68.4%) | 6(31.6%) | |
Middle/Down | 152 | 114(75.0%) | 38(25.0%) | |
TNM stage | | | | 0.021 |
I + II | 95 | 64(67.3%) | 31(32.7%) | |
III | 76 | 63(82.9%) | 13(17.1%) | |
Depth of invasion | | | | 0.008 |
T1 + T2 | 72 | 46(63.8%) | 26(36.2%) | |
T3 + T4 | 99 | 81(81.8%) | 18(18.2%) | |
Lymph node metastasis | | | | 0.047 |
Negative | 79 | 53(67.1%) | 26(32.9%) | |
Positive | 92 | 74(80.4%) | 18(19.6%) | |
Cell culture and reagents
Seven GC cell lines (AGS, BGC823, MGC803, MKN45, MKN1, SGC7901, and HGC27), a gastric mucosa cell line (GES-1), and human kidney cell line (HEK293T) were purchased from GeneChem (Shanghai, China). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO2.
DAPI, Baf-A1, and 3-MA were obtained from Solarbio (Beijing, China). NAC was purchased from Beyotime (Shanghai, China), and 740Y-P (also known as 740YPDGFR) was obtained from MCE (Monmouth Junction, NJ, USA).
Plasmids and cell transfection
FLAG-tagged BDH2, HA-tagged Keap1, and MYC-tagged Nrf2 overexpression plasmid were purchased from GeneChem (Shanghai, China). To construct the BDH2 expression vector, the full-length open reading frame of human BDH2 cDNA was cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen). Expression vectors were transfected into cells with low expression of BDH2 (SGC7901 and BGC823) using Lipofectamine 3000 (Invitrogen). Stably transfected cells were selected for more than 2 weeks using neomycin (G418; Roche, Indianapolis, IN, USA).
Cell proliferation, clonogenic assay, cell cycle analysis, and apoptosis assay
These experiments were performed as described previously [
15].
Immunofluorescence staining
Immunofluorescence staining was performed as described in our previous study [
15]. The cells were incubated with anti-LC3B (1:1000, Abcam, ab48394) and anti-Nrf2 (1:200, Proteintech, 16,396) antibodies at 4 °C overnight. After washing three times with PBS, the cells were stained with secondary antibodies (ABclonal) at 37 °C for 2 h. Finally, Nuclei were labelled with 0.1 g/mL DAPI for 15 min, except when visualising autophagosomes (LC3-II) using an LSM780 laser-scanning confocal microscope (Carl Zeiss). Other images were captured under a BX41 fluorescence microscope (Olympus, Japan).
Transmission electron microscopy (TEM)
As described previously [
15], transfected cells and xenografted tumour tissues were fixed in glutaraldehyde, and then ultrathin sections were prepared with an EM UC7 ultramicrotome (Leica, Solms, Germany). The sections were then imaged by a transmission electron microscope (Hitachi Scientific Instruments, Mountain View, CA, USA) to detect autophagic vacuoles.
Immunohistochemistry (IHC)
The IHC protocol was performed as described previously [
15]. Briefly, tumour sections of patients and nude mouse xenografts were analysed by IHC using the EnVision™ System (Dako, Carpinteria, CA). Primary antibodies used for IHC were anti-BDH2 (1:100, Proteintech, 27,207), anti-Ki67 (1:100, Abcam, ab15580), anti-cleaved caspase-3 (1:200, CST, #9661), anti-LC3B (1:200, Abcam, ab48394), anti-Nrf2 (1:50, Proteintech, 16,396), anti-p-Akt (1:100, Proteintech, 66,444), and anti-p-mTOR (1:100, Abcam, ab109268).
In vivo tumourigenesis assays
Male BALB/c nude mice were randomly divided into two groups (n = 6/group). Two groups of mice were subcutaneously injected with 1 × 107 cells transfected with an empty vector or BDH2 expression vector into the right side of the neck. After tumours were visible, the tumour size was measured every 3 days until 30 days. The formula volume = (length×width^2)/2 cm2 was used to calculate tumour volumes. The mice were sacrificed after 30 days, and the tumour weight was measured. The xenografted tumours were fixed for histological analysis. A TUNEL assay was performed using an In Situ Cell Death Detection Kit (Roche; Mannheim, Germany). The Animal Care Committee of Nantong University reviewed and approved all animal experiments.
Reactive oxygen species (ROS) assay
Measurement of intracellular ROS was performed using 2,7-dichlorofluorescin-diacetate (DCFH-DA) (Yeasen, Shanghai, China). Cells were incubated with 10 μM DCFH-DA diluted in RPMI-1640 medium for 30 min at 37 °C in the dark. The cells were then immediately observed under the fluorescence microscope. Fluorescence intensity was quantified using ImageJ software. For flow cytometry, cells were collected for analysis. For tissue ROS measurement, frozen tumour sections were incubated with DCFH-DA (Item No: E004, Jiancheng Bioengineering, Nanjing, China) at 37 °C for 30 min. The ROS level in the tissue was measured under the BX41 fluorescence microscope.
Autophagic flux analysis
As described previously [
15], cells were transfected with GFP-mRFP-LC3 (Shanghai, China) for 24 h and then treated for 48 h with or without 10 nM Baf-A1. Images were obtained under a confocal microscope (Carl Zeiss).
Quantitative real-time PCR (qRT-PCR)
Total RNA in GC cells was extracted with Trizol reagent (Invitrogen). qRT-PCR was carried out as described previously [
16]. Primer sequences are listed in Table S
1.
Transient transfection and luciferase assay
A pGL3-ARE-Luc reporter was obtained from Genechem, which is a ready-to-transduce ARE responsive lentiviral firefly luciferase reporter to monitor the transcriptional activity of Nrf2. Cells were cultured in 6-well plates and then transfected with the pGL3-ARE-Luc reporter plasmid and pRL-TK plasmid (Promega) using Lipofectamine 3000. A Luciferase Reporter Gene Assay Kit (Beyotime Institute of Biotechnology) was used to determine ARE-driven promoter activity. Luciferase activity is expressed as the ratio to that in control cells.
Coimmunoprecipitation (co-IP)
Total cell lysates were incubated with 1 μg primary antibody or negative control rabbit IgG at 4 °C overnight. Then, 20 μl protein A + G agarose (Bioworld Technology, St. Louis Park, MN, USA) was added, followed by further incubation for 2 h at 4 °C. Protein–antibody complexes were rinsed four times with PBS, and the beads were collected by centrifugation. Proteins were detected by SDS-polyacrylamide gel electrophoresis and western blotting.
Western blotting
Total protein separation and western blotting were performed as described previously [
16]. Western blot analysis was performed using anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9661), anti-p-AMPK (Thr172) (#50081), anti-p-ERK1/2 (#4370), and anti-HA-Tag (#3724) antibodies purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3 (14600–1-AP), anti-P62/SQSTM1 (18420–1-AP), anti-phospho-Akt (Ser-473) (66444–1-Ig), anti-Akt (10176–2-AP), anti-mTOR (20657–1-AP), anti-P38 MAPK (14064–1-AP), anti-JNK (51151–1-AP), anti-ERK1/2 (16443–1-AP), anti-AMPK (10929–2-AP), anti-Nrf2 (16396–1-AP), anti-Keap1 (10503–2-AP), anti-FLAG-tag (66008–2-AP), anti-MYC-tag (60003–2-AP), and anti-β-actin (66009–1-Ig) antibodies were obtained from Proteintech Group Co., Ltd. (Wuhan, China). An anti-phospho-mTOR (Ser-2448) (ab109268) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-p-p38 (sc-7973), anti-p-JNK (sc-6254), and anti-Ub (sc-8017) antibodies were obtained from Santa Cruz Biotechnology., Inc. (Santa Cruz, CA, USA).
Ubiquitination assay
For the polyubiquitinated Nrf2 assay, whole cell lysates prepared with RIPA buffer containing a proteinase inhibitor were subjected to IP of endogenous Nrf2 protein. The levels of Nrf2 ubiquitination were detected by immunoblotting with an anti-Ub antibody.
Assessment of intracellular Iron levels
Iron levels were measured by Iron Colorimetric Assay Kit (ABIN411680, Biovision) according to the manufacturer’s instructions [
13].
Statistical analysis
Data are presented as means ± standard deviation (SD). All experiments were performed at least three times. Statistical analysis was performed with the SPSS software version 22 and GraphPad Prism 7.0 software. The survival rates patients with GC were calculated by Kaplan-Meier and log-rank analyses, and prognostic factors were evaluated using the Cox regression model. Student’s t-test was used for statistical comparisons between experimental groups. p < 0.05 was considered as statistically significant.
Discussion
In this study, we found that BDH2 was significantly down-regulated in GC tissues and that BDH2 expression correlated with adverse clinicopathological parameters in GC. Furthermore, Kaplan–Meier analysis showed that GC patients with low expression of BDH2 had a poor prognosis. Hypermethylation of the gene promoter region is one of the important mechanisms of gene inactivation. We used MethPrimer (
http://www.urogene.org/methprimer/) and did not find any CpG island in the BDH2 promoter region, indicating that the down-regulation or deletion of the BDH2 gene in GC tissues may not be related to DNA promoter methylation. The occurrence of GC is the result of a combination of multiple factors. For example, long-term chronic stimulation of various inflammatory factors is a key factor leading to the occurrence of GC [
23,
24]. Previous studies have shown that sustained inflammation can cause down-regulation of BDH2 expression [
25]. Therefore, we speculate that long-term continuous stimulation of inflammatory factors during the development of GC is an important cause of down-regulation of BDH2 expression in GC tissues.
In the present study, we identified that overexpression of BDH2 inhibited GC cell growth in vitro and in vivo. Iron participates in various biological functions, such as cell proliferation, growth, and ferroptosis [
26]. Evidence from recent studies showed that the mammalian siderophore is an important regulator of cellular iron homeostasis [
27]. Notably, previous studies have shown that biosynthesis of the mammalian siderophore (2,5-DHBA) is catalyzed by BDH2 [
27]. However, whether the biological function induced by BDH2 is related to intracellular iron levels remains unclear. Here, we used a colorimetric assay to measure cytoplasmic iron concentration in GC cells. Our results indicated that overexpression of BDH2 had little effect on iron levels in GC cells (Fig. S
5).
AMPK, MAPK/Erk1/2, and PI3K/Akt/mTOR signalling pathways are classic apoptosis and autophagy pathways [
28‐
30]. Previous studies have shown that BDH2 inhibits HCC cell growth, proliferation, and migration by inducing apoptosis and autophagy [
31]. However, the specific molecular mechanisms of BDH2-induced apoptosis and autophagy remained unclear. Thus, we explored whether BDH2 participates in the regulation of three classic pathways in GC cells. Western blotting indicated that the phosphorylation levels of Akt
Ser473 and mTOR
Ser2448 were downregulated in BDH2-overexpressing cells. However, no significant changes in the expression of p-AMPK, p-p38 MAPK, p-JNK, or p-ERK were found in BDH2-overexpressing cells. Additionally, expression of p-Akt
Ser473 and p-mTOR
Ser2448 was reversed after pretreatment with 740Y-P. Interestingly, pretreatment with 740Y-P also significantly reversed the formation of autophagic flux and enhancement of LC3-II. Moreover, 740Y-P pretreatment reduced BDH2-induced cell death. These results indicated that BDH2 suppressed the growth of GC cells via inhibition of PI3K/Akt/mTOR signalling.
ROS play a key role in the induction of apoptosis and autophagy [
32]. ROS are important signalling molecules in cells, which participate in multiple signalling pathways. A recent report revealed that ROS participate in the induction of caspase-independent cell death in macrophages [
33,
34]. Moreover, ROS induce autophagy and apoptosis of tumour cells by inhibiting pathways such as PI3K/Akt and JNK, thereby suppressing the malignant phenotype of tumour cells [
35,
36]. In this study, a significant increase in ROS generation was detected by flow cytometry and DCFH-DA in the BDH2-overexpressing group, which was clearly inhibited by a ROS scavenger (NAC). We found that BDH2 overexpression triggered apoptosis and autophagy in GC cells by inducing ROS production. However, treatment with NAC drastically rescued apoptotic cell death. In addition, NAC pretreatment remarkably inhibited autophagic flux and conversion of LC3-I to LC3-II. Furthermore, NAC reversed the expression levels of p-Akt
Ser473 and p-mTOR
Ser2448 in BDH2-overexpressing cell lines. Taken together, these results implied that BDH2-induced apoptosis and autophagy were triggered through ROS-mediated PI3K/Akt/mTOR pathways.
ROS produced by oxidative stress are regulated by Nrf2 and its downstream target genes including HO-1 and NQO1 [
37‐
40]. In healthy cells, Nrf2 binds to Keapl in the cytoplasm, which then recruits E3 ubiquitin ligase Cullin 3, thereby facilitating rapid degradation of Nrf2 by proteasomes [
41‐
43]. However, under oxidative stress conditions, Keap1 is uncoupled from Nrf2, allowing Nrf2 to transfer into the nucleus and bind to the ARE, thereby activating downstream antioxidants to increase cellular antioxidant activity. Nrf2 is highly expressed in all kinds of tumour tissues including GC, and cancer cells employ the cytoprotective action of Nrf2 to counter a microenvironment that is not conducive for tumour growth [
44‐
47]. Several mechanisms have been reported for continuous activation of Nrf2 in GC. A nonsynonymous somatic mutation (G333C) was detected in the first repeat of the double glycine repeat/Kelch domain, which does not repress the activity of Nrf2 [
48]. The increased expression of Nrf2 helps to maintain ROS levels below a toxic threshold to escape death in cancer cells [
49]. Recent studies have shown that inhibition of Nrf2 activity suppresses tumour growth and enhances the therapeutic efficiency of chemotherapeutic drugs against cancer [
50‐
53]. Therefore, under different pathological conditions, analysing Nrf2 sheds light on cancer prevention and treatment. In the present study, we found that BDH2 overexpression inhibited ARE-mediated antioxidant gene expression. Moreover, BDH2 downregulated Nrf2 expression through its protein stability rather than transcription. Co-IP verified the effect of BDH2 on binding of Keap1 and Nrf2. Our results suggested that BDH2 stabilises the binding of Keap1 and Nrf2 to promote ubiquitination/degradation of Nrf2 in GC cells.
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