Background
Ovarian carcinoma is one of the leading causes of death among gynecological malignancies [
1] that the most of ovarian carcinoma are firstly detected in advanced stages [
2] and overall 5-year survival rate is as low as 40% [
3]. Current therapeutic strategies against advanced stage ovarian carcinoma include surgical resection along with platinum-based chemotherapy. However, there are serious side effects from platinum-based drugs and surgical intervention [
2]. Therefore, it is critical to understand the molecular events leading to initiation and progression of this devastating disease [
4].
Succinate dehydrogenase (SDH), known as succinate-ubiquinone oxidoreductase, is a mitochondrial enzyme complex that catalyses the oxidation of succinate to fumarate in the citric acid cycle and participates in the electron transport chain [
5]. Mutations of the
SDH gene determine the genetic basis of paragangliomas/pheochromocytomas (PCCs/PGLs) [
6],[
7] and associated tumour syndromes (Carney-Stratakis syndrome and Carney triad) [
8]. Furthermore, mRNA expression of
SDHB is decreased in recovering Ramos cells in childhood non-Hodgkin lymphoma (NHL) [
9]. Reduced SDHB expression is associated with growth and de-differentiation of colorectal cancer cells [
10]. In addition, loss of
SDHB expression is associated with an adverse outcome in PCCs/PGLs, indicating SDHB might be a predictive marker of adverse outcome both in sporadic and familial PCC/PGL [
11]. However, the role of
SDHB in ovarian carcinoma tumourigenesis, especially its association with cellular proliferation, invasion, and migration are not fully elucidated.
Genetic analysis of hereditary paraganglioma reveals an activation of the hypoxia-response pathway [
12]. Immunohistochemical analysis shows strong staining of hypoxia-inducible factor-1α (HIF-1α) and the angiogenic factor, vascular endothelial growth factor in a malignant sporadic pheochromocytoma caused by a germline missense mutation in the
SDHB gene [
13], suggesting that activation of the hypoxia-response pathway may be a common theme underlying SDH (and also FH) mutation [
14],[
15].
Hypoxia-inducible factor-1 (HIF-1), consists of a constitutively expressed β-subunit and an inducib-expressed α-subunit [
16], is a well-established mediator in hypoxia-response pathway. HIF-1α is accumulated under hypoxic conditions, which activates transcription of target genes involved in angiogenesis, energy metabolism, adaptive survival or apoptosis [
17],[
18]. HIF-1α is highly expressed in ovarian cancer and is associated with tumour proliferation [
19], invasion and metastasis [
20],[
21]. AMP-activated protein kinase (AMPK) is a metabolic sensor that helps maintain cellular energy homeostasis and modulate metabolic stresses such as hypoxia and respiratory impairment. AMPK has been linked to the regulation of tumorigenesis [
22] and HIF-1α mediates the growth advantage of tumours with reduced AMPK signaling [
23]. However, the precise linkage between metabolism dysfunction (HIF-1α, AMPK) and the propensity for tumourigenesis has not been fully elucidated in ovarian carcinoma. In current study, we aimed to gain a better understanding of the consequences of
SDHB alteration by gene silencing or overexpression in human ovarian cancer cell lines. Here, we provided the first time that
SDHB silencing resulted in increased tumour cell proliferation, invasion, migration and decreased apoptosis. Conversely, overexpression of
SDHB inhibited cell proliferation, invasion, migration, and promoted apoptosis. Further, HIF-1α and p-AMPKα were found to be upregulated in
SDHB- silenced, but downregulated in
SDHB-overexpressed cancer cells. Moreover, SDHB was downregulated by hypoxia mimetic CoCl2 in human ovarian cancer cells. Our data suggested that SDHB plays an essential role in ovarian cancer cell proliferation, invasion, migration and apoptosis
via AMPK-HIF-1α pathway.
SDHB-overexpression might be a potential therapeutic strategy to inhibit tumour progression in human ovarian carcinoma.
Methods
Tissue specimens and ethics
A total of 25 tissue specimens were collected from surgical patients enrolled in this study, including 7 normal human ovarian epithelium tissues and 18 ovarian carcinoma tissues between July, 2011 to August, 2012. In addition, 7 metastatic ovarian carcinoma were collected. All specimens, obtained during surgery (Department of Obstetrics and Gynaecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, China), frozen immediately in liquid nitrogen and stored at −80°C until analysis. All carcinoma patients were newly diagnosed with ovarian tumours, and received no chemical therapy prior to surgery. The study was approved by the Institutional Review Board of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine. Written informed consents were obtained from all patients. Clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.
Cell lines and cell culture
Human epithelial ovarian adenocarcinoma cancer (EOC) cell lines SKOV3 and A2780 obtained from the Cell Bank Chinese Academy of Science (Shanghai, China), cultured in RPMI1640 medium (Hyclone) supplemented with 10% foetal bovine serum (Gibco). These cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was replaced every 24 h. Hypoxic cells were incubated in the same conditions but in a hypoxic atmosphere with different concentrations of cobalt chloride (CoCl2, Sigma) for 24 h.
Transient transfection of SKOV3 and A2780 cells with SDHB siRNA oligonucleotides
Transient small interfering RNA (siRNA) oligonucleotides were synthesized from Shanghai Integrated Biotech Solutions Co.Ltd. The target sequences were as follows: siSucA
5'-GAT TAA GAA TGA AGT TGA CTC-3'[
24]
, siSucC
5'-GCT CAG AGC TGA ACA TAA TT-3'[
24]. As a control for silencing, we constructed a negative control (NC) siRNA (
5’-UUC UCC GAA CGU GUC ACG UTT-3’) that did not affect
SDHB expression. Transient transfection was carried out using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cells were collected after 24 h for RNA extraction and 48 h for protein electrophoresis.
Transient transfection of ovarian cancer cell SKOV3 with SDHB overexpression pIRES2-EGFP plasmid
The full length SDHB opening reading frame was sub-cloned into pIRES2-EGFP plasmid. The plasmid was purchased from Shanghai Integrated Biotech Solutions Co., Ltd. SKOV3 cells were transfected with the pIRES2-EGFP SDHB vector encoding SDHB cDNA using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Vector-only transfecting cells were used as controls.
RNA extraction and quantitative real-time reverse transcriptase PCR
Total RNA, extracted from cancer cell lines and tissue samples using TRIzol Reagent (Invitrogen, Carlsbad, CA), was reverse transcribed using a PrimeScript RT reagent Kit (TaKaRa, Shanghai, China). Resultant complementary DNAs (cDNAs) underwent quantitative real-time reverse transcriptase PCR using a SYBR Green PCR Master Mix Reagent Kit (TaKaRa). Real-time PCR and data collection were performed on an ABI 7500 real-time system (Applied Biosystems). The primers for SDHB were 5'- AAA TGT GGC CCC ATG GTA TTG-3' (forward) and 5'-AGA GCC ACA GAT GCC TTC TCT G-3' (reverse). The primers for β-actin, serving as the endogenous controls, were 5'-TGA CGT GGA CAT CCG CAA AG-3' (forward) and 5'-CTG GAA GGT GGA CAG CGA GG-3' (reverse). SDS v.1.4 (Applied Biosystems) software was used to perform comparative delta cycle threshold (Ct) analysis. To minimize random variation in cell experiments, each real-time PCR experiment was performed in triplicate.
Succinate dehydrogenase activity assay
Succinate dehydrogenase activity was determined, using succinate dehydrogenase kit (Beijing Solarbio Science & Technology Co., Ltd, China). Protein level was quantified by BCA kit (Cwbiotech, China).
ATP assay
Intracellular ATP concentrations were determined using phosphomolybdic acid colorimetric method (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s protocols. Protein level was quantified by BCA kit (Cwbiotech, China).
Cell proliferation analysis
Cell proliferation was assessed by Cell Counting kit-8 (CCK-8) (Dojindo). Briefly, 1 × 103 SKOV3 and/ or A2780 cells were seeded into 96-well plates treated with SDHB siRNA oligonucleotides or pIRES2-EGFP SDHB vector. After 1, 2, 3, 4, 5 or 6 day treatment, 10 μl CCK8 mixed with 90 μl RPMI1640 was added to each well for further 1.5 h. The absorbance (450 nm) from each well was measured at the indicated time points.
Cell invasion and migration assay
SKOV3 and A2780 cells (6 × 104) (48 h post-transfection) were placed into the upper compartment of the chambers (8 μm pore size, Millipore) coated with 50 μl BD Matrigel (diluted 1:7 in serum-free medium) and placed into 24-well plates. Medium containing 10% foetal bovine serum was added to the lower chamber. After incubation at 37°C for 24 h, cells on the upper side of the membrane were removed using sterile cotton swabs. Cells adhering to the lower surface were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were counted using microscope (Nikon TE300, Tokyo, Japan) at 200× magnification. Five random fields were selected for examination on each membrane, and results were expressed in terms of the invasion cells per field. Each experiment was conducted in triplicate. Cell migration was performed using a similar approach without Matrigel coating, and the treated cells were incubated for 12 h.
Western blot
Cells and tissue samples were lysed in RIPA buffer with PMSF as protease inhibitor (Beyotime, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on NC or PVDF membrane (Millipore, Bedford, MA). After blocking with 5% dry milk in TBST for 2 h at room temperature, the membranes were incubated with the primary antibodies against SDHB (1:1000, Epitomics), β-tubulin (1:4000, Epitomics), β-actin (1:500, Abmart), caspase 3 (1:1000, Cell Signalling Technology), Bcl-2 (1:4000, Epitomics), MMP-2 (1:500, Abcam), FAK (1:1000, CST), p-FAK (1:1000, CST), AMPKα (1:1000,CST), p-AMPKα (1:1000, CST), GAPDH (1:4000, Abmart), P38 (1:1000, CST), p-P38 (1:1000, CST), ERK (1:1000, CST), p-ERK (1:1000, CST), HIF-1α (1:1000, Epitomics) in dilution buffer overnight at 4°C. Membranes were washed for three times with TBST, then were incubated with IRDye 800CW conjugated goat (polyclonal) anti-Rabbit IgG or anti-Mouse IgG (1:10000) antibodies for 1 h at room temperature. The expression of specific proteins was detected through the use of Odyssey system following the manufacturer's instructions.
Statistical analysis
All data were calculated as the means ± standard error (SEM). An independent Student t test was used to compare the continuous variables between groups using GraphPad Prism 5.0 software (San Diego, CA). P < 0.05 was considered statistical significant.
Discussion
In our study, we aimed to gain a better understanding of the role of SDHB in ovarian carcinoma by gene silencing and over expression. SDHB is one of the subunits of SDH takes parts in TCA [
5], ATP is an indicator of energy metabolism. We found that the level ATP was decreased in
SDHB-silenced cancer cells. On the other hand, p-AMPKα and p-P38 MAPK level were increased in the
SDHB-silenced cells, suggesting that
SDHB silencing could activate AMPK pathway in ovarian cancer.
SDHB protein is significantly lower compared with other SDH subunits in colorectal cancer tissues [
10]. Notably, reduced SDHB expression in tumour tissues is associated with tumour de-differentiation. Restoration of SDHB inhibits the growth of cancer cells [
10]. In our current study,
SDHB-silenced cells increased proliferation in SKOV3 and A2780 cells, accompanied with elevated p-ERK level. Bcl-2 is functioned to oppose the apoptosis pathway of programmed cell death [
38],[
39]. Effector caspases are responsible for initiating the events that lead to the hallmarks of apoptosis. Caspase-9, an essential initiator caspase required for apoptosis through mitochondrial pathway, is activated on the apoptosome complex, which directly resulted in cleave and activate effector caspases, such as caspase-3 [
40],[
41]. Anti-apoptotic Bcl-2 level was increased and cleaved caspase 3 was decreased after treated with or without DDP in
SDHB-silenced SKOV3 and A2780 cells.
FAK and MMPs are associated with metastases [
42], secretion and activation of MMP-2 may be responsible for increased motility, invasiveness and metastasis of malignant cells [
43]. In addition, increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis [
44]. We found that
SDHB silencing promoted ovarian cancer invasion and migration accompanied with up-regulated expression of MMP-2 and p-FAK. To explore the role of SDHB in human ovarian carcinoma, we designed plasmid to induce SDHB expression in SKOV3 cells.
SDHB overexpression inhibited cell proliferation in SKOV3. Cellular invasion and migration were inhibited in
SDHB-over-expressed cells accompanied with reduced expression of p-FAK and MMP-2. These observations further verified the role of SDHB in ovarian carcinoma.
The relationship between HIF-1α and SDHB is controversial in tumourigenesis. SDHB is one of the subunits of SDH which takes part in TCA cycle and respiratory chain [
5], AMPK could modulate metabolic stresses such as hypoxia. Study showed mitochondrial dysfunctions could result in reduced HIF-1α protein synthesis through AMPK-dependent manner in HepG2 cells [
37]. Hypoxia is a characteristic of many malignancies arising from various sites [
45] and HIF-1α is a hypoxia responsive factor [
16]. Research reported that HIF-1α was up-regulated in chronically
SDHB-silenced Hep3B cells [
24], HIF-1α was overexpressed in
SDH-deficient leiomyomas and renal cell cancer (HLRCC) [
15]. While there was also report that increased HIF-1α was not associated with loss of SDHB expression on a series of familial and sporadic tumours [
11]. In order to find out the key factor contributed to the phenomenon in our experiment, HIF-1α was analysed in treated cancer cells. The expression of HIF-1α was strongly elevated in
SDHB-silenced ovarian cancer cells. Moreover, SDHB was decreased in CoCl2-treated cancer cells accompanied by HIF-1α up-regulation. In accordance with these results, over-expressed
SDHB could inhibit HIF-1α expression. The results showed
SDHB affected ovarian cancer progression by altering HIF-1α expression. It was suggested that
SDHB silencing up-regulated HIF-1α
via activation of AMPKα in cancers in accordance with the aerobic glycolysis (Warburg effect) [
46]. To verify the effect of
SDHB on energy metabolism in ovarian cancer, p-AMPKα was also analysed in
SDHB-overexpressed SKOV3 cells. The level of p-AMPKα was decreased compared to control. In addition, HIF-1α is decreased by inhibiting ERK pathway in cervical carcinoma CaSki cells [
47], but apoptosis is enhanced by HIF-1α knockdown in pancreatic cancerous BxPC-3 cells [
48]. HIF-1α also promotes cell migration by regulating MMP-2 [
49] and FAK [
50], which is in line with our current data. These results showed SDHB might affect cancer cell proliferation, invasion, migration, and apoptosis
via AMPK-HIF-1α in ovarian carcinoma.
Finally, we compared the expression of SDHB between ovarian carcinoma and normal ovarian epithelium tissues. SDHB mRNA and protein was decreased in human ovarian carcinoma compared with normal ovarian epithelium. In addition, SDHB mRNA expression was decreased in the corresponding metastasis compared with the primary ovarian carcinoma, suggesting SDHB contributed to tumour metastasis. Moreover, it was well known that SDHB-associated PCCs/PGLs often metastasise in a familial setting [
11]. Our current data suggested that SDHB play an important role in cancer progression. Enhanced SDHB expression inhibited cell proliferation, invasion, migration and promoted apoptosis in human ovarian carcinoma. The relationship between SDHB expression and clinical characteristics in human ovarian carcinoma will be determined in our future studies.
Acknowledgements
This work was supported by grants from Shanghai Committee of Science and Technology (Grant No. 10dz2212100, Grant No.12411950200), National Natural Science Foundation (Grant No.81072137, Grant No. 81272882), Shanghai Health Bureau Key Disciplines and Specialties Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LLC, WD and SZ conceived and designed the experiments, LLC and TL performed the experiments, LLC and TL analysed the data, JHZ and YFW contributed reagents/materials/analysis tools, LLC wrote the paper. All authors have approved the final manuscript.