Tumor Biol. (2013) 34:25–31 DOI 10.1007/s13277-012-0506-0
RESEARCH ARTICLE
LDHA is necessary for the tumorigenicity of esophageal squamous cell carcinoma Feng Yao & Tiejun Zhao & Chenxi Zhong & Ji Zhu & Heng Zhao
Received: 7 July 2012 / Accepted: 28 August 2012 / Published online: 8 September 2012 # International Society of Oncology and BioMarkers (ISOBM) 2012
Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal tumors in the world, and the development of new therapeutic targets is needed. Recent studies have shown that aerobic glycolysis, also known as the Warburg effect, mediated the antiapoptotic effects in cancer cells. Lactate dehydrogenase A (LDHA) which executed the final step of aerobic lactate production has been reported to be involved in the tumor progression. However, the function of LDHA in ESCC has not been investigated. In this study, it was found that LDHA was up-regulated in ESCC clinical samples. Knockdown of the expression of LDHA inhibited cell growth and cell migration in vitro as well as tumorigenesis in vivo. With regard to the molecular mechanism, silencing the expression of LDHA was related to decreased AKT activation and cyclin D1 expression and increased cleavage of PARP and caspase 8. Taken together, our findings suggest that LDHA plays an important role in the progression of ESCC by modulating cell growth, and LDHA might be a potential therapeutic target in ESCC.
Feng Yao and Tiejun Zhao contributed equally to this work. F. Yao : C. Zhong : H. Zhao (*) Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, 241 West Huaihai Rd, Shanghai 200030, China e-mail:
[email protected] T. Zhao (*) : J. Zhu Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, 174 Changhai Rd, Shanghai 200433, China e-mail:
[email protected]
Keywords ESCC . LDHA . Cancer metabolism . Cell proliferation
Introduction Esophageal squamous cell carcinoma (ESCC) is the ninth most common malignant tumor and the sixth most common cause of cancer-related death in the world [1]. Despite advances in surgery and chemotherapy, the prognosis of ESCC is very poor [2] . Thus, it is imperative to broaden our knowledge about the mechanisms underlying this cancer and develop novel therapeutic strategy. About 90 years ago, Warburg found that cancer cells displayed high rate of glycolysis even in the presence of oxygen, which was one of the characteristics of tumor cells and hence called Warburg effect or aerobic glycolysis [3]. Lactate dehydrogenase A (LDHA) is one of the subunits of Lactate dehydrogenase, which favors the conversion of pyruvate to lactate. It has long been found that the expression of LDHA was up-regulated in various human cancers [4, 5]. However, the expression and the function of LDHA in ESCC remains unknown. In this study, we investigated the expression and the function of LDHA in the ESCC. It was found that the expression of LDHA was elevated in the clinical ESCC samples. Silencing the expression of LDHA in the ESCC cells inhibited cell growth and migration dramatically. Moreover, down-regulation of LDHA attenuated the tumorigenicity of ESCC cells in vivo. Mechanically, it showed that knockdown of the expression of LDHA activated apoptosis pathway by up-regulation of cleaved PARP and caspase 8 and down-regulation of cyclinD1 and phosphorylated AKT. Taken together, our study revealed the oncogenic role of LDHA in ESCC and suggested that LDHA might be a potential therapy target for ESCC.
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Materials and methods
Cell culture
Primary ESCC Samples
ESCC cell lines Eca109 and Caes17 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) and cultured in DMEM medium (Invitrogen) with 10 % fetal bovine serum, 10 units/ml penicillin G, and 10 mg/ml streptomycin. Cells were incubated at 37 °C in 5 % CO2-humidified air.
Primary tissues were collected from patients who received surgery for ESCC at Shanghai Chest Hospital of Shanghai Jiao Tong University and Changhai Hospital of Second Military Medical University. All patients had given informed consent. Dissected samples were frozen immediately after surgery and stored at >80 °C until needed. Immunohistochemistry Ten-millimeter-thick consecutive sections of ESCC tissues and paired normal tissues were cut and mounted on glass slides. After deparaffinizing, rehydrating, antigen retrieval, and blocking endogenous peroxidases, the sections were washed thrice in 0.01 mol/l PBS (8 mmol/ l Na2HPO4, 2 mmol/l NaH2PO4, and 150 mmol/l NaCl) for 5 min each, blocked for 1 h in 0.01 mol/l PBS supplemented with 0.3 % Triton X-100 and 5 % normal goat serum, followed by an addition of anti-LDHA (1:150) antibody at 4 °C overnight. After brief washes in 0.01 mol/l PBS, sections were exposed for 2 h to 0.01 mol/l PBS containing horseradish peroxidase-conjugated rabbit anti-rabbit IgG (1:500), followed by development with 0.003 % H2O2 and 0.03 % 3,30-diaminobenzidine in 0.05 mol/l Tris–HCl (pH7.6). Immunohistochemistry for each sample was performed at least three separate times, and all sections were counterstained with hematoxylin. The immunohistochemical evaluation for the expression of LDHA in normal tissues and paired ESCC tissues was carried out independently by three pathologists blinded to the patient’s clinical information. German semi-quantitative scoring system was used considering the staining intensity and area extent. Generally, each specimen was assigned a score according to the intensity of the staining (0 0 no staining, 1 0 weak staining, 2 0 moderate staining, and 3 0 strong staining) and the percentage of stained cells (000 %, 101–24 %, 2025–49 %, 3050–74 %, and 4075–100 %). The final immunoreactive score was determined by multiplying the intensity score with the extent of score of stained cells. As a result, nine grades were scored as 0, 1, 2, 3, 4, 6, 8, 9, and 12. “Up-regulation” means that the score for the cancer tissues is higher than the score for the paired normal tissues. “Unchange” means that the score for the cancer tissues is equal to the score for the paired normal tissues. “Downregulation” means that the score for the cancer tissues is lower than the score for the paired normal tissues. When evaluating the intra-tumor expression of LDHA and analyzing the survival, we defined 0–4 score as low and 6–12 as high.
Western blot analysis Western blot analysis was done as previously detailed [1]. Primary antibodies to LDHA, PARP, cyclin D1, caspase 8, and phosphorylated AKT were purchased from Santa Cruz Biotechnology, and antibody to β-actin was purchased from Sigma. Secondary antibodies, rabbit anti-mouse IgG (Sigma) and goat anti-rabbit IgG (Cell Signaling Technology), were used at a dilution of 1:1500. Primary antibodies were diluted in TBST containing 1 % BSA and NaN3. The immunoreactive protein bands were visualized by ECL Kit (Pierce). RNAi-mediated knockdown of LDHA Two target sequences for LDHA small interfering RNA were as listed: 5-ttgttgatgtcatcgaag-3, 5-gggtccttggggaacatg3. The control nucleotide sequence of small interfering RNA was 5-gtacatagggacgtaacg-3, which was the random sequence that was not related to LDHA mRNA. FG12 RNAi vector was used to produce small double-stranded RNA (small interfering RNA) to inhibit target gene expression in ESCC cells. Cell migration assay Boyden chambers (8-μm pore size polycarbonate membrane) were obtained from Neuroprobe Corporation, Bethesda, MD, USA. Cells (2×105) in 0.05-ml medium containing 1 % FBS were placed in the upper chamber, and the lower chamber was loaded with 0.152-ml medium containing 10 % FBS. After 12 h of incubation, the cells that migrated to the lower surface of filters was detected with traditional hematoxylin and eosin staining, and five fields of each well were counted. Three wells were examined for each cell type, and the experiments were repeated for at least three times. Crystal violet assay For cell growth assay, equal number of control cells and cells silencing the expression of LDHA were seeded in sixwell plates and cultured in media supplemented with 10 % FBS for 7 days. Media were changed every other day. Cell growth was stopped after 7 days in culture by removing the medium and adding 0.5 % crystal violet solution in 20 %
Tumor Biol. (2013) 34:25–31
methanol. After staining for 5 min, the fixed cells were washed with phosphate-buffered saline (PBS), photographed, and dissolved with 1 % SDS. The absorbance at 600 nm was evaluated using a micro-plate reader. MTT assay For cell growth analysis, equal numbers of cells were seeded in 48-well plates and cultured for various durations. Cell numbers were measured by MTT assay according to the manufacturer’s protocol (Roche Applied Science).
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subcutaneously injected on the opposite flanks of the same mouse. The resulting tumors were measured once a week, and tumor volumes (in cubic millimeters) were calculated using the standard formula: length×width×height×0.5326. Tumors were harvested 5 weeks after injection and individually weighted. Data were presented as tumor volume (mean ± SD) and tumor weight (mean ± SD). Statistical analysis was performed using the Student’s t test.
Results
Tumorigenicity assay
LDHA was over-expressed in ESCC
Five 4-week-old nude mice were used in this study. LDHA knockdown cells of 1×106 and their control cells were
In order to investigate the expression of LDHA in ESCC samples, the expression of LDHA was examined by western
Fig. 1 Expression of LDHA was elevated in ESCC samples. a Expression of LDHA protein in seven, randomly picked, paired ESCC samples was analyzed by western blot. b Immunohistochemical analysis of LDHA expression in ESCC samples and the matched normal tissues. c LDHA expression scores were shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, respectively; and the vertical bars representing the range of data. We compared the expression of LDHA in cancer tissues and normal tissues using the t test. n0186; double asterisks, P
